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Background: our preliminary data demonstrated that expression and activity of P2X7R was impaired in Systemic Lupus Erythematosus (SLE) and associated with a reduced production of IL-1β1. Serositis is a typical manifestation of SLE characterized by a marked inflammation, which has been suggested to be an “inflammasome driven” manifestation. Objectives: to investigate the role of 1513A>C (rs3751143) and 489C>T (rs208294) Single Nucleotide Polymorphisms (SNPs) which are differently associated with loss or gain of function, respectively, of P2X7R, a potent activator of the NLRP3 inflammasome and IL-1β release, in patients with SLE and with a history of serositis (SLE-S). Methods: DNA was extracted from whole blood and used for evaluation of 2 P2X7R SNPs (1513A>C and 489C>T). Considering the combined action of these two SNPs, the overall activity of P2X7R was divided, into three groups: GOF (gain of function), normal function (NF), and LOF (loss of function). In addition, peripheral blood mononuclear cells (PBMCs) were isolated from venous blood and employed to evaluate P2X7R and NLRP3 expression by RT-PCR, assess P2X7R activity as Benzoyl ATP (BzATP)-induced intracellular Calcium ([Ca2+]i) increments and evaluate in vitro IL-1β following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination. Results: 33 SLE patients (pts), 11 with (SLE-S) and 22 without serositis (SLE-NS) were enrolled. Mean age was 40.9±10.9 years and disease duration was 135.3± 108.6 months. No significant difference in disease activity and clinical characteristic was found between the two groups (table 1). Evaluating 1513A>C SNP, 20 pts were positive for A/A and 13 for A/C phenotype respectively, while in case of 489C>T SNP, 7 pts presented C/C, 12 C/T and 14 T/T phenotype with a comparable distribution between SLE-NS and SLE-S (table 1). After combination of different phenotypes, 9 pts presented normal function (NF), 22 gain of function (GOF) and 2 loss of function (LOF) with no significant difference between SLE-S and SLE-NS (table 1). P2X7R activity, (evaluated as IL-1 β production and [Ca2+]i increments,) and expression (evaluated with RT-PCR) were comparable between SLE-S and SLE-NS. No significant difference was found between expression and activity of P2X7R and NLRP3 and the two SNPs evaluated (table 2). Conclusion: our data indicate that the 1513A>C and 489C>T SNPs do not seem linked with reduced activity and expression of P2X7R that we previously observed in our cohort of lupus patients. Furthermore, no significant association was found between expression of these SNPs and development of serositis. References [1] P2X7 receptor in systemic lupus erythematosus (SLE). Exploring a novel pathogenetic pathway in lupus related serositis. F. Furini. Ann Rheum Dis, volume 77, supplement Suppl, year 2018, page A871 Disclosure of Interests: Federica Furini: None declared, Anna Lisa Giuliani: None declared, Mattia Erminio Parlati: None declared, Marcello Govoni Paid instructor for: Pfizer, Roche, Speakers bureau: Pfizer, Abbvie, MSD, Roche, Eli-Lilly, Celgene, Sanofi, Janssen, Francesco Di Virgilio: None declared, Alessandra Bortoluzzi: None declared

Background Extracellular ATP (eATP) is one of the most diffuse danger associated molecular patterns (DAMPs) released actively through specific mechanisms from intact cells, or passively from damaged or dying cells1. An implication for eATP has been found in SLE2. Among receptors for eATP, P2X7R is deeply involved in inflammatory and immune processes and its activation drives different intracellular pathways such as NLRP3-inflammasome activation, IL-1β maturation and release, IL-6 and TNF-α production, regulation of lymphocyte proliferation and cell apoptosis3. Previous studies pointed out a possible relationship between P2X7R signaling pathways and SLE pathogenesis4 5. A marked inflammatory condition characterize serositis, that are among the most common manifestations of SLE, and chloroquine is one of the main drugs employed. Objectives The aim of this study was to investigate P2X7R expression and activity in SLE. Methods 48 SLE patients, and 20 healthy control (HC) subjects were enrolled. Among SLE patients, 16 (SLE-S) presented, and 32 (SLE-NS) did not present history of serositis. All subjects gave written informed consent to peripheral venous blood withdrawal after approval by the local ethic committee. Plasma samples were used to measure IL-1β, IL-6 and TNF-α levels by ELISA. Mononuclear cells were isolated from blood samples by Ficoll gradient sedimentation and employed as follow: i) assessment of IL-1β, IL-6 and TNF-α release after stimulation with lipopolysaccharide (LPS) and/or Benzoyl ATP (BzATP); ii) evaluation of P2X7R mRNA expression by RT-PCR; iii) measurement of P2X7R activity as BzATP-induced increase of intracellular Ca2+ concentration using the Fura2/AM probe. Results In SLE patients respect to HC, plasma IL-1β levels were unmodified whereas IL-6 was higher, resulting significantly increased in SLE-S. Monocytes isolated from SLE patients released lower quantities of IL-1β after stimulation with BzATP, whereas the release of both IL-6 and TNF-α was significantly augmented in SLE-NS respect to both HC and SLE-S subjects after all types of stimulation. RT-PCR showed reduced P2X7R and augmented NLRP3 mRNA expression in SLE patients. Accordingly, P2X7R activity was significantly reduced in all SLE patients and did not appear to be influenced by a chloroquine pre-treatment. Conclusion In SLE patients, compared to HC subjects, we found reduced P2X7R mRNA expression, increased NLRP3 mRNA, as a possible compensating mechanism, and correspondingly, significantly lower BzATP-induced intracellular Ca2+ increase, without an apparent influence by chloroquine, one of the drugs most diffusely used for SLE treatment. The in vitro IL-1β release was reduced, whereas plasma IL-1β was unaltered, indicating an alternative source, other than monocytes, of this cytokine. Conversely, IL-6 and TNF-α levels were increased in vitro, and IL-6 was present in plasma at higher levels. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 and TNF-α as possible therapeutic targets. References [1] Giuliani, A.L. et al. Extracellular nucleotides and nucleosides as signalling molecules. Immunol Lett, doi:10.1016/j.imlet.2018.11.006 (2018). [2] Magna, M. & Pisetsky, D.S. The Role of Cell Death in the Pathogenesis of SLE: Is Pyroptosis the Missing Link? Scand J Immunol 82, 218-224, (2015). [3] Di Virgilio, F. et al. The P2X7 Receptor in Infection and Inflammation. Immunity 47, 15-31, (2017). [4] Di Virgilio, F. & Giuliani, A.L. Purinergic signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus? Biomed J 39, 326-338, (2016). [5] Faliti, C.E. et al. P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus. J Exp Med, (2019). Disclosure of Interests Anna Lisa Giuliani: None declared, Federica Furini: None declared, Alessandra Bortoluzzi: None declared, Marcello Govoni: None declared, Francesco Di Virgilio Consultant for: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a UK-based biotech Company involved in the development of P2X7R-targeted therapeutics.

Background: Myositis specific antibodies play a central role in the assessment of idiopathic inflammatory myopathies (IIMs) as their presence may indicate specific disease subsets/manifestations or cancer risk. At present, anti-MDA5 antibodies have been associated with the occurrence of rapidly progressive interstitial lung disease (RP-ILD), a life-threatening condition, generally refractory to treatment. Objectives: To describe the relationship between malignancy and anti-MDA5 antibodies in IIMs. Methods: Patients with anti-MDA5+ IIMs and a history of cancer were included in this study. Data were retrospectively collected. Because of the lack of RP-ILD shared definitions, we defined RP-ILD as an admission to the Intensive Care Unit (ICU) for IIMs related respiratory insufficiency. Results: In our cohort (n=133, 67% females) we identified 17 patients (13%) with a history of cancer (8 males [47%], 9 females [53%]; median age at IIMs onset 62 years [IQR 53-67], at cancer diagnosis 56 years [IQR 51-66]). All but 1 patient had solid tumours, 5 (29%) had breast cancer. In 9 patients (53%) cancer diagnosis ranged ± 3 years from IIMs onset; in 3 patients the delay exceeded 120 months. Three patients (18%) developed RP-ILD respectively 82, 66 and 0 months after the diagnosis of cancer. Four (24%) patients died respectively 0 (neoplasia), 0 (RP-ILD in ICU, diagnosis of cancer after an autopsy), 7 (neoplasia) and 66 (infection after RP-ILD in ICU) months after cancer diagnosis. The 13 surviving patients had a median rheumatological follow-up of 47 months (IQR 19-84) and an oncological follow-up of 57 months (IQR 10-130). Interestingly, in the overall cohort 66% of patients without cancer (76 out of 116) had less than 3 years of follow-up. Of the 22 RP-ILD patients without neoplasia 14 died, 11 during ICU admission. In the latter patients, we could not rule out an occult neoplasia due to the emergency of the situation. Only one patient, with a 18 month history of IIMs before ICU admission, underwent autopsy and was reported negative for cancer. Conclusion: This is the first study addressed to tackle the relationship between cancer and anti-MDA5+ IIMs. Although in some cases no clear relationship between IIMs and cancer can be proved because of the time gap, in others the timing of occurrence of cancer and IIMs was very close, suggesting a link between these manifestations. Finally, we emphasize the importance of an accurate neoplasm screening in anti-MDA5+ RP-ILD, because treatment refractoriness could suggest the paraneoplastic nature of the manifestation. References [1] Barsotti S, et al. Rheumatol Int 2014 [2] Lu X, et al. PLoS One 2014 Disclosure of Interests: Ludovico De Stefano: None declared, Santos Castaneda Consultant for: Amgen, BMS, Pfizer, Lilly, MSD, Roche, Sanofi, UCB, Ellen De Langhe: None declared, Jorg Distler: None declared, Johannes Knitza: None declared, Ilaria Cavazzana: None declared, alain meyer: None declared, Jorge Rojas-Serrano: None declared, eugenio arrigoni: None declared, Federica Furini: None declared, Marcello Govoni Paid instructor for: Pfizer, Roche, Speakers bureau: Pfizer, Abbvie, MSD, Roche, Eli-Lilly, Celgene, Sanofi, Janssen, paola parrocchini: None declared, Giovanni Zanframundo: None declared, Jose Antonio P. da Silva: None declared, elisa pedrollo: None declared, Florenzo Iannone Consultant for: F Iannone has received consultancy fees and/or speaker honoraria from Pfizer, AbbVie, MSD, BMS, Novartis, Lilly, UCB outside this work, Speakers bureau: F Iannone has received consultancy fees and/or speaker honoraria from Pfizer, AbbVie, MSD, BMS, Novartis, Lilly, UCB outside this work, Carlomaurizio Montecucco Speakers bureau: AbbVie, Bristol-Myers Squibb, Celgene, Sanofi, Genzyme, Lilly, MSD, Pfizer, UCB, Lorenzo Cavagna: None declared

Background Diffuse alveolar haemorrhage (DAH) is a rare and severe manifestation of ANCA-associated vasculitides (AAV). Objectives To identify predictors of survival in patients with AAV-DAH. Methods A retrospective study of 102 consecutive patients (50% females; mean age 59±17 years) from 27 Italian Centres diagnosed with AAV-DAH was planned. Cox regression unadjusted analyses were performed. Results Among AAV patients, 47% had Granulomatosis with Polyangiitis (GPA), 47% Microscopic Polyangiitis (MPA) and 6% Eosinophilic Granulomatosis with Polyangiitis (EGPA). At DAH onset, mean BVAS was 20±8 and most patients had renal involvement (RI). Admission to Intensive Care Unit was needed in 27% of patients, while ventilatory support (VS) was required in 46%. At least one cardiovascular risk factor (CVRF) was recorded in 48%. Over a median follow-up of 39 months (25%–75% IQR 66 months), 19/102 patients (18.6%) died (figure 1). All patients received high-dose glucocorticoids in association with Cyclophosphamide (CYC 78%, mean cumulative dose 8±7 g) or Rituximab (37%). Plasma exchange was performed in 46%. Infections occurred in 38%. Age>65 years (HR 3.05 [95% CI 1.18–7.9], p=0.04), CVRF ≥2 (HR 8.85 [95% CI [AG7] 2.34–33.50], p=0.01), BVAS (v.3) (HR 1.07 [95% CI 1.01–1.13], p=0.01) were associated with mortality, whereas FFS was not. The need for VS (HR 4.54 [95% CI 1.48–13.85], p=0.008) and infections (HR 3.98 [95% CI 1.48–10.69] were also associated with mortality. Conclusions Older age, VS, CVRF and infections affect the survival in AAV. There is a need for specific outcome measeures. Disclosure of Interest None declared

Background Anti-MDA5 antibodies are a known set of antibodies observed mainly in dermatomyositis, typically associated with cutaneous involvement, presence and often rapid progression (RP) of interstitial lung disease (ILD) and amyopathic muscle involvement. Despite the increased attention, described cohorts involves only a limited number of cases Objectives to define clinical characteristics of a large cohort of anti-MDA5 antibodies positive patients Methods Retrospective assessment of anti-MDA5 positive patients from centres referring to our group Results 82 anti-MDA5 positive cases (56 females) were collected. In median: onset age was 44 Y (IQR 22.57), diagnostic delay 4 Mo (IQR 1–10), follow-up 13 Mo (IQR 3–43). Fifty-three patients had ILD, that was RP in 24 cases (15 at ILD onset, 9 after ILD onset). Fifthteen ILD patients were admitted in Intensive Care Unit (ICU): 4 were treated with Extracorporeal-Membrane-Oxygenation, 7 with mechanical-invasive and 2 with mechanical-non-invasive ventilation. Forthy-nine patients had muscle involvement (39 symptomatic), 42 arthritis, 54 cutaneous involvement, 9 history of malignancy. Twelve patients died (ILD=7, ILD and sepsis=3, neoplasia=1, not specified=1), 7 of those in ICU Conclusions In our cohort, ILD was the most frequent finding. A RP of ILD was common, occurring also in ILD with symptomatic/chronic onset. The low rate of survival in ICU raised the problem of follow up and early treatment of ILD. In our cohort arthritis was common and muscle involvement mainly symptomatic. Finally, the high percentage of observed malignancies suggests a careful neoplastic screening and follow up Reference [1] Labrador-Horrillo M, et al. J Immunol Res2014;2014:290797. Disclosure of Interest None declared

Background Antisynthetase syndrome (ASSD) is an autoimmune disease characterised by the clinical triad arthritis, myositis, and interstitial lung disease (ILD). Despite Raynaud’s phenomenon (RP) is another typical feature of ASSD, nailfold videocapillaroscopy (NVC) assessment of these patients has been only sporadically described, without the elucidating data for clinicians. Objectives To describe NVC features of ASSD patients and to investigate possible correlations with clinical and serological features of the disease. Methods We retrospectively analysed NVC images of 190 ASSD patients (females/males 3.76, mean age 49.7±12.8 years, mean disease duration 51.2±71.4 months, 133 anti-Jo-1 and 57 non-anti-Jo-1 positive patients). For each patient, we examined number of capillaries, giant capillaries, micro-haemorrhages, avascular areas, ramified capillaries, and the presence of scleroderma (SSc) patterns. Finally, we correlated NVC features with clinical and serological findings of ASSD patients. Results NVC abnormalities were observed in 62.1% of AASD patients compared with 29.3% of a group of 75 patients with primary Raynaud’s phenomenon (p Conclusions NVC abnormalities are commonly observed in ASSD, independently to the occurrence of RP. The presence of a SSc-like pattern should let to identify a more defined ASSD subtype and prospective studies could confirm the association with clinical and serological features of ASSD. Disclosure of Interest None declared

Background Antisynthetase syndrome (ASSD) is an autoimmune disease characterized by the clinical triad arthritis, myositis, and interstitial lung disease (ILD). As in inflammatory myopathies, nailfold videocapillaroscopy (NVC) alterations have been sporadically described also in ASSD patients, but no elucidating data are available. Objectives To investigate the possible specific NVC features of ASSD patients. Methods Within the framework of a multicenter study, we retrospectively analyzed NVC images of ASSD patients, after excluding patients with overlap syndrome with systemic sclerosis. Two operators in a blind manner re-evaluated all patients with at least one image per finger. For each patient, we examined number of capillaries (mean number of capillaries per mm in the distal row), enlarged and giant capillaries, micro-hemorrhages, avascular areas, ramified capillaries, and the presence of a scleroderma (SSc)-like pattern, according to Manfredi et al. Finally, we correlated NVC features with clinical and serological findings of ASSD patients. Results The NVC of 54 ASSD patients were analyzed (males/females 1/6.8, mean age 55.79, CI95% 51.9–59.9 years, mean disease duration 59.4, CI95% 27.9–90.9 months). Raynaud9s phenomenon (RP) was recorded in 51.9% of patients, arthritis in 79.6%, myositis in 53.7%, and ILD in 92.6%. NVC alterations were observed in 53.7% of AASD patients. Nineteen patients (35.2%) showed a SSc-like pattern; the main features were disarrangement of hairpin and angiogenetic aspects (42.6%), avascular areas (38.9%), giant capillaries (27.6%), and microhemorrhages (20.4%). Finally, the mean number of capillaries was reduced (7.8±2/mm). No significant association was recorded between SSc-like pattern and the presence of arthritis, myositis, and ILD, nor with RP. Among other NVC features, angiogenesis was significantly associated to female gender (p=0.031), while microhemorrhages were inversely associated to the presence of arthritis (0.033). No association was observed between NVC features and autoantibodies profile. Of interest, in 58% of patients with ILD we observed at least a NVC alteration vs no patients without ILD (p=0.04). Finally, in patients with RP NVC alterations were recorded in 15/28 patients (53.6%) and a SSc-like pattern in 11/28 (39.3%), while only 57.9% of patients with SSc-like pattern had a clinically manifest Raynaud9s phenomenon. Conclusions Despite preliminary, the present is the first study concerning NVC in AASD patients. Regardless of the presence of Raynaud9s phenomenon, NVC alterations are frequently observed; in particular, a SSc-like pattern is recorded in more than 1/3 of patients. NVC should be performed in all ASSD patients at diagnosis regardless of the presence of RP in the patient history and during follow-up. ASSD should be always considered in the screening of RP. A prospective multicenter study has been planned to identify specific patterns and possible associations between NVC findings and clinical and serological features of ASSD. References Manfredi A, et al. 2015. Sebastiani M, et al. 2014. Disclosure of Interest None declared

Background The occurrence of osteoporosis (OP) and fragility fractures (FF) is a frequent comorbidity in patients with systemic lupus erythematosus (SLE), mainly due to the concomitant presence of many risks factors for secondary osteoporosis (drugs, gender, concomitant diseases). The use of corticosteroids (CS) increases the risk of osteoporosis induced by corticosteroid (GIOP) and FF [1]. Denosumab is monoclonal antibody that binds to RANKL, inhibiting osteoclast formation and activation, used for both male and women OP to prevent FFx [2,3]. Previous RCT [2] reported a numerically higher serious adverse events of infections in the denosumab users. In particular severe skin infections were significantly more frequent [4]. The possible increase in infection risk might be a concern in subjects treated with CS. The use of denosumab in SLE with GIOP patients is recently reported in 17 cases within a RCT [5]. Anyway, no data regarding the tolerability and disease activity during therapy were reported Objectives Our aim was to analyze the prevalence, the tolerability, and relevant changes in disease activity or damage in a cohort of SLE patients treated with denosumab Methods among all SLE patients currently followed in two referral Center we selected the ones that received at least one dose of denosumab. Clinical, serological manifestations, concomitant diseases and therapies were collected from clinical charts. Damage index (SDI), activity index (SLEDAI-2K) were calculated when denosumab was introduced (first cycle) and at the last evaluation (last cycle). For statistical analysis Chi-squared test or Fisher exact test was used Results Among 793 patients in our cohorts, 21 (2.6%) were treated with denosumab. Demographic data reported a female prevalence (19 cases,90%), mean age at onset of 38±12years, mean duration of disease of 28±10years and mean follow-up of 21±7.8years; most frequent manifestations were arthritis (90%), cutaneous (67%) and neurological (67%). All patients were still treated with CS (mean duration of 25±6.8 years) with a daily dose of 8.5±3.5mg/day. Other risk factors for OP were present in the majority of them: drugs (anticoagulants in 6pts, cyclosporin in 3 and antiepileptic in 2), chronic kidney disease (CKD) (4pts), hypovitaminosis D (15pts), anorexia, celiac disease and hemochromatosis (1pt, each). Indication for denosumab was OP with FFx in 17 cases: denosumab was used for a new FF during biphosphonates or after teriparatide. In the other 4 the indication was primary prevention with contraindications at the use of biphosphonates for concomitant severe CKD. Mean duration therapy was 4 cycles (range 1–8): data regarding disease activity, damage and adverse events are reported in table 1. No new FFx developed in any of the included patients at the last evaluation available. Conclusions In our cohorts denosumab is still used in few selected patients. However it could be considered as a valid option in GIOP patients because it was globally well tolerated and in our cohort it9s efficacy in the prevention of new fragility fractures was confirmed References Gudbjornsson, et al. Ann Rheum Dis 2002;61,32–36. Cummings SR et al. N Eng J Med 2009; 61:756–65. Austin M et al. J Bone Miner Res 2012;27:687–93. Watts NB, Osteoporos Int 2012;23:327–37. Mok CC, et al. Bone 2015;75:222–8. Disclosure of Interest None declared