M. Pendolino

Citrullination and autoimmunity

Autoimmune diseases are characterized by the bodys own immune system attack to the self-tissues, a condition enabled, in predisposed subjects, by the reduction of self-tolerance. A central role has been recently recognized to post-translational modifications, since they can promote generation of neo-(auto)antigens and in turn an autoimmune response. During the last years great attention has been paid to citrullination, because of its role in inducing anti-citrullinated proteins/peptide antibodies (ACPA), a class of autoantibodies with diagnostic, predictive and prognostic value for Rheumatoid Arthritis (RA). Nonetheless, citrullination has been reported to be a process present in a wide range of inflammatory tissues. Indeed, citrullinated proteins have been detected also in other inflammatory arthritides and in inflammatory conditions other than arthritides (polymyositis, inflammatory bowel disease and chronic tonsillitis). Moreover, environmental exposure to cigarette smoke and nanomaterials of air pollution may be able to induce citrullination in lung cells prior to any detectable onset of inflammatory responses, suggesting that protein citrullination could be considered as a sign of early cellular damage. Accordingly, citrullination seems to be implicated in all those para-physiological processes, such as cells death pathways, in which intracellular calcium concentration raises to higher levels than in physiologic conditions: hence, peptidylarginine deiminases enzymes are activated during apoptosis, autophagy and NETosis, processes which are well-known to be implicated in autoimmunity. Taken together, these data support the hypothesis that rather than being a disease-dependent process, citrullination is an inflammatory-dependent condition that plays a central role in autoimmune diseases.

New autoantigens in the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent miscarriages or fetal loss, and circulating antiphospholipid antibodies (aPL). Enzyme-linked immunosorbent assays for anticardiolipin and anti-β2-glycoprotein I antibodies and clotting assays for the lupus anticoagulant are the tests recommended for detecting aPL. However, the aPL are a heterogeneous group of antibodies directed against anionic phospholipids but also toward phospholipid-binding plasma proteins or phospholipid-protein complexes. β2-glycoprotein I (β2GPI) is the playmaker antigen of APS, however during apoptosis, lysophospholipids can become exposed on the cell surface, and mainly through their interaction with β2GPI, they can become targets of aPL. Some CL metabolites are likely to escape from the remodeling cycle. This would account for the progressive loss of mitochondrial CL during apoptosis, as well as for the presence of CL and lyso-CL at the cell surface, where they can interact with β2GPI and become targets of aPL. Other recognized targets of aPL are represented by phosphatidylserine, lyso(bis)phosphatidic acid, Phosphatidylethanolamine, vimentin, and annexin A5. These molecules may allow improving the knowledge on the pathogenesis, and the early identification of APS. Although several studies have shown the presence of antibodies directed against other antigens in APS, their clinical relevance is still a matter of debate, and it needs to be confirmed with experimental data and longitudinal studies.

IL-18 Serum Level in Adult Onset Still’s Disease: A Marker of Disease Activity

Introduction. Immunological factors seem to play a pivotal role in Adult Onset Stills Disease (AOSD). Among all, IL-18 cytokine is overexpressed and drives the inflammatory process. Objective. We aimed to investigate the levels of IL-18 in sera of Italian patients with AOSD and to assess its possible role as a marker of disease activity. Methods. IL-18 serum levels were determined by ELISA in 26 Italian patients with AOSD. Disease activity was assessed using Pouchots criteria. As controls, 21 patients with Rheumatoid Arthritis (RA), 21 patients with Sjogrens Syndrome (SS), 20 patients with Systemic Lupus Erythematosus (SLE), and 21 healthy subjects (normal human sera, NHS) were evaluated. Results. IL-18 serum levels were significantly higher in patients with active AOSD than in non-active (P = 0.001) and control groups (RA P = 0.0070, SS P = 0.0029, SLE P = 0.0032, NHS P = 0.0004). A significant correlation between IL-18 serum levels and disease activity (P < 0.0001), and laboratory parameters as ferritin (P = 0.0127) and C-reactive protein (P = 0.0032) was demonstrated. Conclusions. Higher levels of IL-18 are detected in active AODS patients and correlate with disease activity and inflammatory laboratory features. ROC-AUC analysis of the serum concentration of IL-18 suggests that it can be considered a diagnostic marker of AOSD. This paper supports the targeting of this cytokine as a possible therapeutic option in AOSD.

International consensus: What else can we do to improve diagnosis and therapeutic strategies in patients affected by autoimmune rheumatic diseases (rheumatoid arthritis, spondyloarthritides, systemic sclerosis, systemic lupus erythematosus, antiphospholipid syndrome and Sjogren's syndrome)?: The unmet needs and the clinical grey zone in autoimmune disease management

Autoimmune diseases are a complex set of diseases characterized by immune system activation and, although many progresses have been done in the last 15years, several unmet needs in the management of these patients may be still identified. Recently, a panel of international Experts, divided in different working groups according to their clinical and scientific expertise, were asked to identify, debate and formulate a list of key unmet needs within the field of rheumatology, serving as a roadmap for research as well as support for clinicians. After a systematic review of the literature, the results and the discussions from each working group were summarised in different statements. Due to the differences among the diseases and their heterogeneity, a large number of statements was produced and voted by the Experts to reach a consensus in a plenary session. At all the steps of this process, including the initial discussions by the steering committee, the identification of the unmet needs, the expansion of the working group and finally the development of statements, a large agreement was attained. This work confirmed that several unmet needs may be identified and despite the development of new therapeutic strategies as well as a better understanding of the effects of existing therapies, many open questions still remain in this field, suggesting a research agenda for the future and specific clinical suggestions which may allow physicians to better manage those clinical conditions still lacking of scientific clarity.

Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases

BackgroundAntibodies against carbamylated proteins (anti-CarP) have been recently identified in the sera of patients with rheumatoid arthritis (RA). The objective of the study was to evaluate the prevalence, sensitivity and specificity of anti-CarP compared to anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF), replicating the existing data in a large cohort of Italian patients with RA and extending the evaluation to other autoimmune rheumatic diseases (AIRDs).MethodsSerum samples (n = 607) from 309 patients with RA, 200 disease controls and 98 normal healthy subjects (NHS) were evaluated. Anti-CarP were detected using carbamylated fetal calf serum as the antigen. ACPAs were detected using second-generation ELISA and IgM RF was assessed as part of routine analysis.ResultsAnti-CarP antibodies were detected in 117 patients with RA (34.4%), ACPA in 190 patients (61.4%) and RF in 202 patients (65.3%). Two (2.04%) of the NHS were positive for anti-CarP, one NHS (1.02%) was positive for ACPA and three NHS were positive for RF (3.06%). Among disease controls, anti-CarP antibodies were detected in 33 patients (16.5%), ACPA in 29 patients (14.5%) and RF in 64 patients (32%). In particular, 16.8% of patients with systemic lupus erythematosus and 31.1% of patients with Sjögren syndrome were positive for anti-CarP. The sensitivity of anti-CarP, ACPA and RF was 46.8%, 61.8% and 64.4%, respectively and specificity was 91.95%, 89.93% and 76.51%, respectively.ConclusionsThe present study extends the knowledge of anti-CarP antibodies, confirming previous data on the diagnostic accuracy of anti-CarP in RA in a large cohort of Italian patients. Anti-CarP antibodies demonstrated relatively low sensitivity and slightly higher specificity compared to ACPA and RF. Even if predominantly present in RA, anti-CarP was detected in a variable percentage of patients with other autoimmune rheumatic diseases and their generation could be attributed to the inflammatory status; the clinical relevance of anti-CarP antibodies in these latter patients should be further determined.

Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes

T lymphocytes from patients with Systemic Lupus Erythematosus (SLE) display multiple abnormalities, including increased cell activation, abnormal cell death by apoptosis and impairment of autophagy pathway. In the present study we report the presence of specific antibodies to D4GDI, a small GTPase family inhibitor, in a significant percentage (46%) of SLE patient sera. We also found a significant association between the presence of these autoantibodies and hematologic manifestations occurring in these patients. Investigating the possible implication of anti-D4GDI autoantibodies in SLE pathogenesis or progression, we found that these antibodies were capable of binding D4GDI expressed at the lymphocyte surface and triggering a series of subcellular events, including Rho GTPase activation. These antibodies were also able to induce autophagy in T cells from both healthy donors and SLE patients, but only those negative to these antibodies. We can conclude that anti-D4GDI autoantibodies could be capable of triggering important responses in T cells such as cytoskeleton remodeling and autophagy pathway and that, in SLE patients, the chronic exposure to these specific autoantibodies could lead to the selection of autophagy-resistant T cell clones contributing to the pathogenesis of the disease.

SAT0378 Autophagy is Up-Regulated in the Salivary Glands of Primary Sjogren's Syndrome Patients and Correlates with the Focus Score and Disease Activity

Background Autophagy is now considered as a major regulator in trafficking events that activates innate and adaptive immunity and consistent evidence supports its role in autoimmunity (1). Primary Sjogrens syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B cells that, producing chemokines and cytokines, coordinate the chronic inflammatory process. No data on the role of autophagy in pSS are available in humans, although studies in mice demonstrated its involvement in the salivary and lacrimal gland homeostasis (2,3). Objectives We investigated the autophagy process in salivary gland tissue and in peripheral T lymphocytes from pSS patients to evaluate its possible implication in the pathogenesis of the disease. Methods 30 patients with pSS, 20 patients with sicca syndrome or non-specific-chronic-sialoadenitis and 30 healthy donors were studied. Peripheral T lymphocytes were isolated by standard procedures. Salivary gland biopsies were evaluated by i) H&E to assess histological pattern, the severity of inflammatory infiltrate and the presence of germinal centers, ii) RT-PCR for the expression of autophagy-related genes and IL-23p19 and IL-21 mRNA. Autophagy-related proteins (LC3, Atg5, p62/SQSTM1) were detected in peripheral T lymphocytes by western blot and in salivary gland by immunohistochemistry and immunofluorescence. IL-21 and IL-23p19 serum levels were measured by ELISA. Results Autophagy is up-regulated in T cells from the salivary glands, but not from the peripheral blood, of pSS patients and it is correlated with disease activity and damage indexes. Autophagy is also correlated with the local expression of the pro-inflammatory cytokines IL-21 and IL-23p19, but not with serum levels of these cytokines. Conclusions Our data show that, in pSS, T cells present high levels of autophagy, which may up-regulate the expression of pro-inflammatory cytokines, providing evidence for a role of this process in the pathogenesis of pSS and identifying a possible therapeutic target. References Pierdominici M, Vomero M, Barbati C et al. FASEB J. 2012; 26: 1400-1412. Morgan-Bathke M, Lin HH, Chibly AM et al. J Dent Res. 2013; 92: 911-917. Seo Y, Ji YW, Lee SM, et al. Cell Death Dis. 2014; 5: e1309. Disclosure of Interest None declared

THU0696 Anti-carbamylated proteins antibodies in sle patients with joint involvement: a possible new biomarker for erosive damage

Background The traditional concept of non-erosive arthritis in Systemic Lupus Erythematosus (SLE) patients changed during the last years, thanks to the use of more sensitive imaging techniques, such as ultrasonography (US). The predictive role of Rheumatoid Arthritis (RA)-specific autoantibodies for bone loss in SLE patients has been investigated. In particular, anti-citrullinated peptide antibodies (ACPA) have been identified in about 50% of SLE patients with x-Ray detected erosive arthritis. More recently, anti-carbamylated proteins (anti-CarP) antibodies have been demonstrated in seronegative RA patients, with a prevalence of about 16% and a significant association with erosive damage. Objectives In the present cross-sectional study, we aimed at assessing the association between anti-CarP antibodies and erosive damage in a large cohort of SLE patients with joint involvement. Methods For this purpose, we evaluated 152 SLE patients (1997 ACR criteria; M/F 11/141, mean±SD age 46.4±11.3 years, mean±SD disease duration 144.9±110.5 months) with joint involvement (arthralgia/arthritis). The clinical and laboratory data were collected in a standardized computerized electronically filled form. All patients underwent blood draws to detect Rheumatoid Factor (RF) and ACPA, by using commercial ELISA kits according to the manufacturers instructions, and anti-CarP antibodies by home-made ELISA (results were expressed in arbitrary units (AU)/ml and values above 340 IU/ml were considered positive). US was performed to assess the bone surfaces of metacarpophalangeal and proximal interphalangeal. At each joint, according with OMERACT definition, the presence of erosions was registered with a dichotomous value (0/1), allowing the possibility to obtain a total score, ranging from 0 to 20. Results The prevalence of anti-CarP antibodies was 28.3%, similar to RF (27.6%) and significantly higher to ACPA (11.2%, p=0.003). The mean±SD titer of anti-CarP antibodies was 890.5±794.9 IU/ml. Thirty-nine patients (25.6%) showed erosive arthritis: all the patients referred the occurrence of at least one episode of clinical synovitis. Erosive arthritis was significantly associated with anti-CarP antibodies (p=0.004) and ACPA (p=0.0008). Moreover, a significant correlation between anti-CarP antibody titer and US-erosive score was observed (r=0.2, p=0.01). Of note, anti-CarP antibodies were identified in 24.5% of double negative (ACPA-/RF-) patients, with erosive damage in 25% of them. Interestingly, anti-CarP antibodies resulted significantly associated with the presence of anti-dsDNA (P=0.01). Conclusions In the present study, for the first time, we identified a significant association between anti-CarP antibodies and erosive damage in SLE-related arthritis, in terms of frequency and severity. We found these antibodies in about 25% of ACPA-/RF- SLE patients, a prevalence higher than that described in seronegative RA patients. Taken together, our results suggest that anti-CarP antibodies could be considered as a candidate biomarker of severity in SLE patients with joint involvement. Disclosure of Interest None declared

FRI0028 In vitro inhibitory effect of etanercept on autophagy: a new mechanism of action of tnf inhibitors in rheumatoid arthritis

Background Autophagy has emerged as a key mechanism in the development, survival and function of immune cells and dysregulation of autophagic pathway has been implicated in the pathogenesis of several autoimmune diseases including Rheumatoid Arthritis (RA) (1). In fact, autophagy seems to be involved in the generation of citrullinated peptides, with consequent breakage of tolerance in RA (2). Moreover, increased autophagy levels and a reduction of apoptosis-related molecules have been found in RA synovial tissues and a role of TNF-induced autophagy in RA development has been proposed (3). Objectives The aim of the study was to analysed the effect of TNF and anti-TNF inhibitor etanercept on autophagy and apoptosis in cells involved in RA pathogenesis. Methods Peripheral blood mononuclear cells (PBMCs) and fibroblast-like synoviocytes (FLS) isolated from RA patients were cultured in presence of TNF and in serum deprivation state (starvation) for 4 hours and then etanercept, at concentration of 15 ug/mL, were added to the culture. After 24h cells were analyzed for levels of autophagy marker LC3-II by western blot and for percentage of annexin V-positive apoptotic cells by flow cytometry. Results As expected, TNF and starvation induced autophagy on RA PBMC and FLS in dose-dependent manner after 24h of culture (p<0.05 in all experimental conditions). Moreover, the adding of etanercept caused a significant reduction of LC3-II levels (p=0.004) and an increase of apoptosis rate (p=0.002) after both pro-autophagic stimuli (p<0.05). Conclusions We demonstrated for the first time an inhibitory effect of etanercept on autophagy activation of cells involved in RA pathogenesis. In addition, our findings suggest a crucial role of autophagy in RA cells survival. References Pierdominici M, Vomero M, Barbati C et al. Role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus. FASEB J. 2012; 26:1400–12. Sorice M, Iannuccelli C, Manganelli V et al. Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies. Rheumatology. 2016;55:1374–85. Rockel JS, Kapoor M. Autophagy: controlling cell fate in rheumatic diseases. Nat Rev Rheumatol. 2016;12:517–31. Disclosure of Interest None declared

FRI0328 A Complete Remission Lasting at Least One Year Influences The Outcome in Patients Affected by SLE: Results from A Large Monocentric Cohort

Background The achievement of complete remission is one of the most important goal in the management of patients with Systemic Lupus Erythematosus (SLE). Even though a definition of remission has been proposed (complete absence of any clinical and serological sign or symptom of activity) and the concomitant acceptable treatment (including antimalarial drugs only) has been identified, however the minimum lapse of time is still controversial. This issue is of primary importance in a disease characterized by a relapsing-remitting course. Several cut-off have been suggested, ranging from 1 to 5 years. Objectives The primary end-point of the present study was to analyze the frequency of complete remission lasting at least 1 year in a large monocentric SLE cohort. Furthermore, we aimed at evaluating its association with different clinical and serological parameters and its impact on the chronic damage accrual. Methods Data on Caucasian SLE patients, diagnosed according to the ACR Classification Criteria for SLE, were collected. We included in the present analysis SLE patients evaluated at least twice per year during the last 5 years. The frequency of complete remission, defined as an SLE Disease Activity Index 2000 (SLEDAI-2k) =0, was assessed in glucocorticoid-free and immunosuppressant-free patients. Antimalarial drugs were the only SLE-related acceptable treatment. Results Our database includes 658 SLE patients evaluated at least once. From these patients non-Caucasian were excluded, resulting in 622 patients. Among these, 179 (M/F 13/166, mean age 46.3±12.7 years, mean disease duration 173.9±101.9 months) fulfilled the above-reported selection criteria. During the 5-year follow-up, 27 patients (15.1%) experienced a complete remission lasting for at least 1 year, with a mean duration of 37.5±15.8 months. Notably, six patients experienced a prolonged complete remission lasting 5 years (4.6%). There were no significant differences in terms of demographic characteristics between patients achieving and those not achieving remission. Patients in remission showed a significantly lower frequency of renal involvement (29.0 vs 18.4, P=0.04) and low C3 and C4 (14.8% vs 36.8%, P=0.0005; 14.8% vs 33.5%, P=0.0002, respectively). There were no other differences in terms of clinical and laboratory features. The frequency and the severity of chronic damage, evaluated by SLICC Damage Index (SDI), resulted significantly lower in patients achieving remission (11.1% vs 51.9%, P<0.000001; 0.48±0.8 vs 0.8±1.2, P=0.03, respectively). The length of the remission status did not influence the chronic damage. At the multivariate analysis, no independent factors were associated with remission; conversely, low C3 resulted a risk factor for absence of remission (OR=0.15, 95% CI 0.03–0.06). Conclusions In the present SLE cohort, a complete remission lasting at least 1 year has been identified in 15% of patients. The remission status, regardless of its duration, is associated with a lower chronic damage, in terms of frequency and severity. These results suggest that the achievement of a state of remission lasting at least one year may represent a favorable prognostic factor in patients with SLE. Disclosure of Interest None declared