T. Colasanti

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

Diesel exhaust particles induce autophagy and citrullination in Normal Human Bronchial Epithelial cells

A variety of environmental agents has been found to influence the development of autoimmune diseases; in particular, the studies investigating the potential association of systemic autoimmune rheumatic diseases with environmental micro and nano-particulate matter are very few and contradictory. In this study, the role of diesel exhaust particles (DEPs), one of the most important components of environment particulate matter, emitted from Euro 4 and Euro 5 engines in altering the Normal Human Bronchial Epithelial (NHBE) cell biological activity was evaluated. NHBE cells were exposed in vitro to Euro 4 and Euro 5 particle carbon core, sampled upstream of the typical emission after-treatment systems (diesel oxidation catalyst and diesel particulate filter), whose surfaces have been washed from well-assessed harmful species, as polycyclic aromatic hydrocarbons (PAHs) to: (1) investigate their specific capacity to affect cell viability (flow cytometry); (2) stimulate the production of the pro-inflammatory cytokine IL-18 (Enzyme-Linked ImmunoSorbent Assay -ELISA-); (3) verify their specific ability to induce autophagy and elicit protein citrullination and peptidyl arginine deiminase (PAD) activity (confocal laser scanning microscopy, immunoprecipitation, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis -SDS-PAGE- and Western blot, ELISA). In this study we demonstrated, for the first time, that both Euro 4 and Euro 5 carbon particles, deprived of PAHs possibly adsorbed on the soot surface, were able to: (1) significantly affect cell viability, inducing autophagy, apoptosis and necrosis; (2) stimulate the release of the pro-inflammatory cytokine IL-18; (3) elicit protein citrullination and PAD activity in NHBE cells. In particular, Euro 5 DEPs seem to have a more marked effect with respect to Euro 4 DEPs.

AB0163 Increased eryptosis levels in erythrocytes treated with antibodies from aps patients: eryptosis as new actor in aps pathogenesis

Background Erythrocytes (RBCs) are highly sensitive cells constantly exposed to several stress stimuli including inflammatory mediators. Despite the absence of nuclei and the lack of crucial elements in the machinery of apoptosis, they have developed a rapid self-destruction process called eryptosis. During this process the externalization of phosphatidylserine (PS) activates the correct elimination of erythrocytes by phagocytes preventing inflammation and intravascular hemolysis. It has been recently demonstrated that PS-exposing erythrocytes are able to adhere to endothelial cells causing an impairment of circulation,1 suggesting a possible involvement of eryptosis in the increased risk of thrombotic episodes typical of antiphospholipid syndrome (APS). Objectives Enhanced eryptosis is known to contribute to several pathological conditions but the role of this process in APS has not been investigated yet. For this reason, we evaluated the effect of antibodies (Abs) purified from APS patients and healthy subjects positive for antiphospolipid antibodies without clinical manifestations (aPL carriers) on eryptosis activation. Moreover, spontaneous eryptosis levels in APS, aPL carriers, autoimmune haemolytic anaemia (AIHA) and healthy donors (HD) were analyzed. Methods 30 patients with primary APS (M/F 7/23, mean age 50.5±8.2 years), 17 aPL carriers (M/F 4/13, mean age 48.6±8.3 years) were recruited after written informed consent. Moreover 13 AIHA patients and 17 HD were also enrolled as positive and negative control group respectively. Ammonium sulfate precipitation is used to purify Abs from sera of APS and aPL carriers subjects. RBCs, isolated from whole blood by centrifugation, were incubated with APS and aPL carriers Abs at concentration of 20ug/mL and after 4 hours the percentage of annexin V-positive cells (PS-exposing cells) was analyzed by flow cytometry. The same technique was used to estimate spontaneous eryptosis levels in all cohorts studied. Results In vitro Abs from APS induced eryptosis in RBCs isolated from HD after 4 hours of culture compared to untreated and RBCs stimulated with Intravenous Immunoglobulins (IVIG), both p= 0.02. On the contrary, Abs from aPL carriers had no effect on the percentage of PS-exposing RBCs (figure 1). Ex vivo, APS patients showed higher levels of spontaneous eryptosis compared to HD (p=0.001). As expected, eryptosis was upregulated in AIHA patients compared to all populations studied (p<0.0001). Interestingly, the percentage of annexin V- positive RBCs was lower in aPL carriers respect to APS patients (p=0.001). No significant correlation between eryptosis and clinical parameters was found.Abstract AB0163 – Figure 1 Conclusions In this study we demonstrated a new aspect of APS pathogenesis based on the capacity of Abs isolated from APS patients, and not those from aPL carriers, to stimulate eryptosis suggesting a possible contribution of this process in APS clinical manifestations. Reference [1] Borst O, Abed M, Alesutan I, et al. Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX. Am J Physiol Cell Physiol 2012;302:C644–51. Disclosure of Interest None declared

OP0257 Decrease of autophagy in peripheral blood mononuclear cells from systemic lupus erythematosus patients treated with belimumab

Background Autophagy is a conserved catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, promoting the recycling of cellular nutrients, and is also a key mechanism for protein homeostasis and quality control1. T lymphocytes from patients with systemic lupus erythematosus (SLE) are resistant to induction of autophagy2. Belimumab (BLM), a human monoclonal antibody that inhibits B lymphocyte stimulator (BLyS), is the first biological drug to be approved for the treatment of SLE. BLM seems to play a role in modulating the signalling cascade involved in the regulation of autophagy, blocking the binding of soluble BLyS to its receptors (B cell activating factor receptor, BAFF-R; B cell maturation antigen, BCMA; transmembrane activator and calcium modulator and cyclophilin ligand interactor, TACI), mainly expressed on B cells and plasmacells3. Objectives The aim of this study was to evaluate the autophagy process by means the expression of LC3-II and p62 markers in lysates of peripheral blood mononuclear cells (PBMCs) from SLE patients at baseline (t0) and after 2 weeks (t2weeks), 1 month (t1month), and 3 months (t3months) of treatment with BLM. We also investigated the presence of BLyS receptors on T cell subsets. Methods We enrolled 15 consecutive patients who started treatment with BLM (M/F, 0/15; mean age, 44.3 years, range 30–54 years; mean disease duration, 242.6 months, range 48–432 months). All patients fulfilled the American College of Rheumatology revised classification criteria4. PBMCs from SLE patients were lysed in lysis buffer and analysed to evaluate autophagy, monitoring LC3-II and p62 levels by Western blot. Flow cytometry was performed for surface phenotyping of freshly isolated PBMCs, using conjugated monoclonal antibodies against human CD4 and CD8; anti-human BAFF-R, BCMA, and TACI polyclonal antibodies were used to detect BLyS receptors on T cells subsets. Results LC3-II expression levels in PBMCs from SLE patients decreased after 3 months of BLM therapy and, in the same lapse, p62 levels increased (figure 1; p<0.05 for all the experimental conditions). BAFF-R and BCMA were expressed on CD4+ (Mean Fluorescence Intensity -fold increase-, MFI=1.6 and 1.2, respectively; p<0.05) and CD8+ (MFI=1.6 and 2.5; p<0.05) T cells, while TACI was expressed only on CD8+ T cells (MFI=1.2; p<0.05). Conclusions In the present study we demonstrated, for the first time, the expression of BAFF-R, TACI and BCMA in CD4+ and CD8+ T cells from SLE patients, and that BLM treatment was able to decrease the levels of autophagy in PBMCs. We can speculate that BLM could mediate this effect by blocking the binding of BLyS with its receptors. References [1] Kaur, Debnath. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol2015;16:461–72. [2] Alessandri, et al. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy. FASEB J2012;26:4722–32. [3] Rockel, Kapoor. Autophagy: controlling cell fate in rheumatic diseases. Nat Rev Rheumatol2016;12:517–31. [4] Hochberg. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum1997;40:1725. Disclosure of Interest None declared

Citrullination and Autophagy

During evolution, the eukaryotic cells develop different processes in order to adapt themselves to environmental changes, as well as to die or to survive. These processes include apoptosis, NETosis, and autophagy: citrullination is implicated in all of these physiological mechanisms.

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

AB1170 Prevalence of anti-carp antibodies in patients with primary sjÖgren's syndrome: association with clinical, serological and histological aspects

Background The presence of antibodies against carbamylated peptides (anti-CarP) has been associated with increased disease activity and severe joint damage in rheumatoid arthritis. Our group has demonstrated their presence also in other inflammatory conditions with a high prevalence in Sjogrens Syndrome (SS) (14/45, 31.1%)1. There is only one study up to now investigating anti-CarP in SS demonstrating a prevalence of these antibodies in 27% of cases and an association with the presence of germinal centres (GCs)2. Thus, these antibodies have been proposed as a new tool for identifying SS patients with a more aggressive disease. Objectives To confirm the presence of anti-CarP antibodies in a large monocentric cohort of patients with SS and investigate their association with clinical, serological and histological features. Methods Serum samples from consecutive patients with SS (AECC criteria) were collected and stored at -20°. Anti-CarP antibodies were detected by a modified solid-phase “home-made” ELISA1. The mean +3 times SD was used as cut-off. Minor paraffin embedded salivary glands were stained by H&E and IHC using lymphocytes T and B markers [anti-CD3, anti-CD20 (DAKO)]. GCs presence was defined by H&E and confirmed by identification of follicular dendritic cells (anti-CD21, DAKO). Images were analysed as follows: focus score (FS) calculation, mean foci area, presence of segregated foci (SF), GCs and lymphoepithelial lesions (LELs). Results Clinical and laboratory features of SS patients are shown in table. Serum anti-CarP were detected in 30/104 patients (28.8%) without association with any clinical or serological feature (Fishers exact test). Positive patients were more likely to present SF (P=0.024). No association was found with the presence of GCs or LELs. Anti-CarP titre correlated with the FS (P=0.045, r=0.304), the number of foci (P=0.008, r=0.347), mean foci area (P=0.028, r=) and the percentage of SF (P=0.046, r=0.331) (Spearmans test). Prevalence of anti-CarP was higher in patients with arthritis [7/15 (46.6%)] than those without ([23/89 (25.8%)] (p>0.05). Mean anti-CarP titre was higher in patients with arthritis compared to those without (322.3±173.8 aU/ml vs 279.5±171.1, P=0.004, respectively). Conclusions This is the largest cohort of SS patients screened for anti-CarP so far. Our results show a prevalence of anti-Carp antibodies in agreement with the literature; however, no association was found with any clinical or serological aspect. Anti-CarP do not seem to be associated with histological features predictive of lymphoma, i.e. CGs and LELs. Nonetheless, considering the titre correlation with the FS, mean foci area and percentage of segregation, higher serum levels of anti-CarP may reflect a severe tissue inflammation more prone to form organized infiltrates. This finding, in association with the evidence of higher levels in patients with arthritis, may support the idea that these antibodies are useful to measure the severity of systemic inflammation. References Pecani A et al. Arthritis Res Ther. 2016 25;18(1):276. Bergum B et al. Ann Rheum Dis. 2016;75(8):1494–500. Disclosure of Interest None declared

FRI0027 Tnf expression on microparticles from rheumatoid arthritis patients mediates endothelial cell fate in vitro

Background Microparticles (MPs) are small membrane vesicles released by many cell types under physiological conditions or pathological situations. Increased levels of MPs have been reported in patients with autoimmune diseases, such as Rheumatoid Arthritis (RA) (1) which is characterized by an accelerated atherosclerosis. TNF, a key cytokine involved in the pathogenesis of RA, has been associated to RA atherosclerosis (2). Moreover, MPs could also have a role in endothelial dysfunction contributing to atherosclerosis in RA patients. Objectives The aim of this study was: 1) to evaluate TNF expression on RA-MPs. 2) to estimate the effects of serum RA-MPs on endothelial apoptosis and autophagy before and after in vivo and in vitro treatment with Etanercept. Methods 15 RA patients were recruited from the Department of Rheumatology Sapienza University of Rome at baseline (T0) and after three months of therapy (T3) with Etanercept. A fasting blood sample was collected and centrifuged two times to obtain platelet-poor plasma rich in MPs. The resulting plasma was stained with Ab anti-TNF and analized by flow cytometry. In vitro effects of serum RA-MPs on endothelium were evaluated using human umbilical vein cell line EA.hy926. Cells were treated with RA-MPs purified at T0 and T3 and with RA-MPs in vitro treated with Etanercept. At the end of experiments apoptosis and autophagy were evaluated. Apoptosis was analyzed by flow cytometry using a FITC-conjugated annexin V and propidium iodide apoptosis detection kit; autophagy was analyzed by western blot for the expression level of the autophagic marker LC3-II. Results Our results showed that MPs purified from RA patients at T0 expressed TNF on their surface and this expression decreased after three months of treatment with Etanercept (p=0.04). Moreover, serum RA-MPs at T0 significantly increased, in a dose-dependent manner, both apoptosis and autophagy levels in the human umbilical vein cell line EA. hy926 (p=0.005 and p=0.02, respectively versus untreated cells). After three months of treatment with Etanercept, RA-MPs were not able to significantly change these parameters.Finally, in vitro studies showed that RA-MPs treated with Etanercept significantly decreased surface expression of TNF and were no longer able to modulate apoptosis and autophagy in EA.hy926 cells. Conclusions Our data demonstrate that serum RA-MPs express TNF on their surface. Moreover, both in vivo and in vitro treatment with Etanercept interfere with the ability of MPs to significantly modulate apoptosis and autophagy of endothelial cells by decreasing surface expression of TNF. References Beyer C and Pisetsky DS.The role of microparticles in the pathogenesis of rheumatic diseases. Nat. Rev. Rheumatol. 2010;6:21–9. Feldmann M, Brennan FM, Foxwell BM and Maini RN.The role of TNF alpha and IL-1 in rheumatoid arthritis Curr. Dir. Autoimmun. 2001;3:188–99. 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