C. Barbati

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.

The role of dietary sodium intake on the modulation of T helper 17 cells and regulatory T cells in patients with rheumatoid arthritis and systemic lupus erythematosus

We aimed at investigating whether the frequency and function of T helper 17 (Th17) and regulatory T cells (Treg) are affected by a restriction of dietary sodium intake in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We enrolled RA and SLE patients not receiving drugs known to increase urinary sodium excretion. Patients underwent a dietary regimen starting with a restricted daily sodium intake followed by a normal-sodium daily intake. The timepoints were identified at baseline (T0), after 3 weeks of low-sodium dietary regimen (T3), after 2 weeks of normal-sodium dietary regimen (T5). On these visits, we measured the 24-hour urinary sodium excretion, the frequency and function of Th17 and Treg cells in the peripheral blood, the serum levels of cytokines. Analysis of urinary sodium excretion confirmed adherence to the dietary regimen. In RA patients, a trend toward a reduction in the frequencies of Th17 cells over the low-sodium dietary regimen followed by an increase at T5 was observed, while Treg cells exhibited the opposite trend. SLE patients showed a progressive reduction in the percentage of Th17 cells that reached a significance at T5 compared to T0 (p = 0.01) and an increase in the percentage of Treg cells following the low-sodium dietary regimen at both T1 and T3 compared to T0 (p = 0.04 and p = 0.02, respectively). No significant apoptosis or proliferation modulation was found. In RA patients, we found a reduction at T5 compared to T0 in serum levels of both TGFβ (p = 0.0016) and IL-9 (p = 0.0007); serum IL-9 levels were also reduced in SLE patients at T5 with respect to T0 (p = 0.03). This is the first study investigating the effects of dietary sodium intake on adaptive immunity. Based on the results, we hypothesize that a restricted sodium dietary intake may dampen the inflammatory response in RA and SLE patients.

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

PS2:24 Plasmatic and urinary endothelial microparticles are increased in patients with lupus nephritis

Systemic lupus erythematosus(SLE) is a chronic autoimmune disease,characterised by alterations in both the innate and adaptive immune system ultimately leading to the loss of immunologic tolerance and occurrence of autoantibodies against nuclear material. Lupus nephritis is one of the most severe features of SLE determining an increase in morbidity e mortality rates.Renal biopsy still represent a fundamental diagnostic and prognostic tool for LN.Therefore, non-invasive surrogate biomarkers of active LN are urgently needed.Circulating, heterogeneous subcellular microparticles(MPs) are released from cells and platelets constitutively and upon cellular activation or apoptosis.Such MPs may reflect the state of their parental cells and tissues, and could serve as markers of pathology. Particularly in SLE, MPs are potential biomarkers and triggers of autoimmunity.Recent studies have demonstrated increased of plasmatic EMPs in patients with SLE active disease and their reduction after treatment. The aim of this study was to investigate levels of EMPs in a cohort of SLE patients with and without renal involvement compared to healthy controls. MPs were isolated from plasma and urine and characterised by flow cytometry using AnnessinV (a probe that binds to the exposed phosphatidilserine – PS)and antibodies against surface markers endothelial cells(CD31 +CD41-). Sixty SLE patients and 29HC were studied. Twenty-eight patients had renal involvement. The total number of plasmatic MPs was lower in SLE patients than HC(p=0.001). In contrast there was no significant difference EMPs between the two groups. When the patients were divided according to renal involvement, the patients with active-LN(A-LN) showed lower plasmatic EMPs in comparison to inactive LN(I-LN) (p=0.01), while the patients with I-LN had higher EMPs than HC(p=0.002).There was no significant difference of total urinaryMPs between SLE patients and HC. UrinaryEMPs were higher in SLE and in LN patients than HC. The results of the present study show increased EMPs in patients with LN in remission. Circulating-EMPs have been considered as a potential biomarker of endothelial activation and damage in several autoimmune disorders, and higher EMP have been detected in patients with vasculitis and associated with disease activation. According to our results, plasmatic EMPs are higher in inactive-LN patients than in HC. These results may suggest a potential role of EMP as a biomarker of LN.

PS2:33 Microparticles from sle patients are a source of interferon-alpha

Systemic lupus erythematosus (SLE) is the prototype of systemic autoimmune disorders. Interferon alpha is a pleiotropic cytokine that can affect multiple cell types involved in lupus. Dendritic cells (DC) have a special role in the production of IFN and are the main sources of serum interferon. IFN has the potential to dramatically influence the development, progression, and pathogenesis of SLE as it can influence the function and activation state of most major immune cell subsets and function as a bridge between innate and adaptive immunity. Circulating microparticles (MPs) are ubiquitous in the blood of healthy individuals, These MPs play an active role in coagulation and intercellular communication and assist in activation or suppression of the immune system, depending on their parental cell origin. Changes in the concentration and/or composition of circulating Mps have been described in various autoimmune diseases, including rheumatoid arthritis (RA) systemic sclerosis (SSc) and SLE. For SLE, the reported microparticle-related changes remain somewhat inconclusive. To better understand the role of MPs in SLE patients, we analysed the presence of IFN-alpha on MPs surface. MPs were isolated from citrate-treated plasma; blood cells were removed by two steps of centrifugation process (2500 g for 15 min at 20 C two time). The resulting platelet-poor-plasma (PPP), was analysed by flow cytometry with specific antibody against IFN-alpha 20 consecutive SLE patients (10 with active lupus nephritis) and 10 sex- and age-matched healthy control subjects (HC) were included in the study. We found that MPs from SLE patients carry on their surface IFN alpha. Moreover, the percentage IFNalpha +MPs was higher in SLE patients and in lupus nephritis patients than in HC, but there was not significant difference between patients with and without renal involvement. The results of the present study show for the first time the presence of IFN-alpha on MPs surface. We may assume that INF+MPs derive from DC. In lupus nephritis patients the increased recruitment of DC was at tubular interstitial level, with subsequent IFN-alpha production. Interestingly, MPs (containing RNA and DNA) could stimulate type I IFN production in plasmacytoid dendritic cells and subsequently the release of MPs from DC.

PS7:136 Apoptotic effect of blys on endothelial cells and endothelial progenitor cells is mediated by blys receptors and is reverted by belimumab

Circulating endothelial progenitor cells (EPCs) are markers of endothelial function; their reduction and functional impairment in patients with Systemic Lupus Erythematosus (SLE), partially account for endothelial dysfunction. In murine models of atherosclerosis, treatment with a B Lymphocyte Stimulator (BLyS) reduced atherosclerotic plaque size and progression. In a case study on SLE 20 women, our group confirmed a decrease in the number of EPCs, with a significant increase after treatment with Belimumab (BLM). The aims of this study were: to evaluate the ex vivo and in vitro effects of BLyS stimulation and inhibition on the EPC colonies and on endothelial cells; to investigate BLyS receptor expression of on EPCs and endothelial cells. EPCs were isolated from peripheral blood mononuclear cells and defined as CD34+/VEGF-R2 +double positive cells. To evaluate the ability to form colonies, the EPCs of 2 SLE patients and 2 healthy controls were cultured on fibronectin-coated dishes and incubated with BlyS or Blys and BLM and counted after 7 days. Apoptosis of EPCs and endothelial cell line (EA.hy926) was evaluated after 6, 12 and 24 hours incubation with BLyS and after 6 hour with BLyS and BLM. EPC and EA.hy926 were also incubated with anti-B activating factor-receptor (BAFF-R) antibodies, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), then analysed by cytofluorimetry. The number of EPC colonies in patients was lower than in controls; moreover, colonies were poorly organised compared to controls; BLM incubation restored the structure of the colonies. After 6 hours of incubation, BLyS (20 ng/ml) induced apoptosis of EPC and EA.hy926; co-incubation with BLM inhibited the apoptotic effect. Both EPCs and EA.hy926 expressed BAFF-R (MFI=3.8 and 1.5 respectively) and BCMA (MFI=1.25 and 1.15); EPCs also express TACI (MFI=1.4). The results of this study showed: a quantitative and qualitative alteration of colonies in patients, restored after ex vivo and in vitro BLM treatment; the apoptotic effect of BLyS on EPC and endothelial cells inhibit by BLM and the preferential expression of BAFF–R on the surface of EPC and EA.hy926.

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

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