Search

ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS) patients.MethodsSalivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production.ResultsTranscriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α).ConclusionsOverall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

Background:Systemic sclerosis (SSc) is an inflammatory connective tissue disease leading to chronic and progressive fibrosis, typically affecting the skin and internal organs. The alteration of both innate and adaptive immune responses plays a pivotal role in SSc pathophysiology, although it has not yet been fully elucidated [1].Recent findings have demonstrated interleukin (IL)-9 overexpression and significant group 2 innate lymphoid cells (ILC2) expansion in patients with SSc. Th9-ILC2-mast cells axis seems to be involved in SSc tissue damage and in the induction of fibrosis [2]. Activation and production of IL-9 by Th9 cells are promoted by transforming growth factor (TGF)-β, thymic stromal lymphopoietin (TSLP), IL-25 and IL-33. Thus, the IL-25 / IL-17RB pathway would act as a key player in SSc.Objectives:The purpose of this study was to evaluate the role of the IL-25 / IL-17RB axis as a driver in Th9 polarization and ILC2 expansion and polarization in SSc patients.Methods:26 patients were enrolled in this study. Peripheral blood and skin biopsy specimens were obtained from SSc patients. PBMCs were isolated and incubated with and without recombinant (r)IL-25 for 24-48-72 hours and the frequencies of Th9 cells, Th17 cells and ILC2 were assessed by flow cytometry analysis. Moreover, the ex vivo expression of IL-17RB in ILC2 was also assessed. Immunofluorescence analysis was performed on biopsy skin samples to evaluate IL-17RB expression in ILC2.Results:In SSc samples, Th9 cells frequency progressively increased after stimulation with rIL-25, compared to healthy controls in which IL-9 frequency decreased over time regardless of rIL-25.Simultaneously, we evaluated the role of the IL-25 / IL-17RB axis in Th17 cells.In the SSc pool, the initially low rate of IL-17 increased at 72 hours after stimulation with rIL-25. In unstimulated SSc samples, the initially higher IL-17 rate decreased at 72 hours; conversely, it was consistently low in healthy controls, at both baseline and stimulated conditions.Our results confirmed the presence of IL-25-dependent clonal ILC2 expansion, suggesting a greater and progressive expansion over time in patients with SSc, compared to controls.Interestingly, increased IL-17RB expression was found in circulating ILC2 from SSc patients supporting the characterization of ILC2 inflammatory phenotype.Consistently, immunofluorescence on the skin of SSc patients showed a marked infiltrate of CD3-GATA3+ IL-17RB+ cells, confirming the presence of the activated inflammatory phenotype ILC2, absent in skin biopsies of healthy controls (Figure 1).Conclusion:These preliminary data suggest an active role of the IL-25/IL-17RB axis in SSc. It results in Th9 polarization and Th17 clonal expansion, inducing the production of IL-9 and, to a lesser extent, IL-17. Moreover, in addition to promoting Th9-mediated ILC2 differentiation, IL-25 directs the polarization of ILC2 towards the inflammatory phenotype.References:[1]Denton CP, & Khanna D. (2017). Systemic sclerosis. Lancet (London, England), 390(10103), 1685–1699.[2]Guggino G, Ciccia F, Di Liberto D, Lo Pizzo M, Ruscitti P, Cipriani P, Ferrante A, Sireci G, Dieli F, Fourniè JJ, Giacomelli R, Triolo G. s.l. Interleukin-9 over-expression and T helper 9 polarization in systemic sclerosis patients. Clin Exp Immunol, 2016 Dec.Figure 1.Immunofluorescence on biopsy skin samples of SSc patients (top) and healthy control (bottom).Disclosure of Interests:Lidia La Barbera: None declared, Marianna Lo Pizzo: None declared, Diana Di Liberto: None declared, Claudia Schinocca: None declared, Piero Ruscitti Consultant of: Pfizer, Novartis, Roche, Lilly, Celgene, Abbvie, Rorberto Giacomelli Consultant of: Pfizer, Novartis, Roche, Lilly, Celgene, Abbvie, Francesco Dieli: None declared, francesco ciccia Consultant of: Pfizer, Novartis, Roche, Lilly, Celgene, Abbvie, Giuliana Guggino Consultant of: Pfizer, Novartis, Roche, Lilly, Celgene, Abbvie

Dear Editor, We read with interest the correspondence by Chen et al 1 about our recent article on the comparison of clinical, laboratory and artificial intelligence–radiological findings in patients with lung involvement either from macrophage activation syndrome (MAS), a secondary form of haemophagocytic lymphohistiocytosis (HLH) or severe coronavirus disease 2019 (COVID-19).2 Age is one of the most common confounding factors in any observational study since it is associated with an increased risk of comorbidities, which may influence the outcome. In our study, the matching for age was not reliable because of higher prevalence of severe COVID-19 in elderly patients, who were admitted to intensive or subintensive care units of our hospital. These results mirror what was already observed in other observational studies,3 4 in which the incidence and severity of COVID-19 are generally higher in elderly patients due to higher frequency of comorbidities, increased frailty and immunosenescence.5 Conversely, MAS complicating adult-onset Still’s disease (AOSD), as patients assessed in our study, usually affects young adults.6 Considering the scientific debate behind our study,2 about the possibility that severe COVID-19 could be considered or not part of HLH spectrum, the age of occurrence may further differentiate the clinical pictures between these diseases. Furthermore, Chen et al 1 questioned the use of Yamaguchi criteria in classifying AOSD patients .7 Although …

Background:Rheumatoid arthritis (RA) manifests as a chronic, symmetrical articular disease; any synovial joint may be involved and the cervical spine is another possible affected site [1,2]. Although the cervical spine involvement is a very severe complication of RA, the exact incidence of cervical spine abnormalities in RA is not fully elucidated yet.Objectives:In this work, we aimed at evaluating the incidence of a cervical spine involvement and associated clinical characteristics in patients with RA in a single centre observational study.Methods:In this study, we performed a retrospective analysis of prospectively followed-up patients with RA attending our Rheumatologic Clinic, University of L’Aquila, L’Aquila, Italy, between January 2010 and December 2020. Patients who met the diagnostic criteria of 1987 ACR and/or ACR/EULAR 2010 criteria were assessed. Each patient was investigated for cervical spine involvement occurrence, particularly the atlas-axis cervical involvement. Instrumental imaging evaluation of the cervical spine was performed in all patients with clinical suspicion of involvement, by both MRI and CT examinations. MRI examinations were performed on a 1.5 Tesla scanner, acquiring T1, T2, and STIR sequences on sagittal, coronal and axial planes. CT examinations were performed on a multidetector 320-row CT scanner (Aquilion One,Toshiba) acquired with a thin collimation; soft tissue or bone algorithms were applied for image data reconstruction and analysis.Incident cases of cervical spine involvement were reported as incidence proportion and incidence rate per 1000 person-years at risk.Results:In this evaluation, 347 consecutive patients, (mostly female 87.6%, mean age of 66.1 ± 11.3 years) attending our Rheumatologic Clinic, were studied. The incidence proportion of cervical spine involvement was 2.0% [0.6 – 3.4%], occurring in 7 out of 347 patients, and identified by both MRI and CT scan. Considering over 1623 person-years, an incidence rate of 4.0 × 1000 [3.0 – 5.0] person-years was also estimated. All our patients with cervical spine involvement were characterised by female gender, rheumatoid factor, ACPA, fulfilment of ACR 1987 classificative criteria, and poorly controlled disease activity. Smoking habit and extra-articular manifestations were also frequently reported. Glucocorticoids, methotrexate, and infliximab were mainly administered in these patients. This therapeutic strategy resulted in a complete resolution of the inflammatory synovitis in all but one patient. The latter developed a cervical spinal instability resulting in neurological progression and required a subsequent surgical management.Conclusion:The cervical spine involvement is an uncommon but serious manifestation of RA, since possibly causing severe neurologic damage. A poorly controlled inflammatory process would be an important risk factor for developing a cervical spine involvement in RA, thus, a proper management is required, from an early accurate recognition to a timely therapeutic strategy. MRI could provide a useful clinical tool to early evaluate the cervical spine involvement in RA. Finally, considering the low incidence, further studies are needed to fully estimate the exact incidence of this manifestation, mostly in poorly symptomatic patients.References:[1]Shlobin NA, Dahdaleh NS. Cervical spine manifestations of rheumatoid arthritis: a review. Neurosurg Rev. 2020 Oct 10. Neurosurg Rev. 2020 Oct 10. doi: 10.1007/s10143-020-01412-1.[2]Kothe, R.Rheumatoid instability in the cervical spine: Diagnostic and therapeutic strategies. Orthopade. 2018 Jun;47(6):489-495.Disclosure of Interests:None declared

Background:Adult-onset Still’s disease (AOSD) is a rare systemic inflammatory disorder usually affecting young adults, burdened by life-threatening complications, mainly macrophage activation syndrome (MAS), a secondary form of hemophagocytic lymphohistiocytosis [1]. In this context, the importance of an accurate assessment of AOSD is suggested to promptly recognise the multivisceral involvement of the disease which is associated with life-threatening complications. The assessment of the most aggressive subsets of the disease could guide the clinicians when to apply additional resources but avoiding unnecessary expenditures in patients with a less severe clinical picture.Objectives:In this study, we aimed at describing the multivisceral involvement of the disease to retrieve imaging-based differences in AOSD patients with and without MAS.Methods:The present evaluation has been designed as a cross-sectional study to descriptively compare the multivisceral involvement in AOSD patients with and without MAS. Patients admitted to our Institution, who underwent a total body CT scan, were selected from our historical cohort and assessed. Clinical and CT scan characteristics of AOSD patients with and without MAS were compared. Clinical and CT scan characteristics of AOSD patients with and without MAS were analysed by parametric or non-parametric t tests for all continuous variables, and chi squared test was used for categorical ones, as appropriate. Furthermore, possible correlations among radiological outcomes with laboratory markers and systemic score were estimated by using a point-biserial coefficient correlation.Results:This study evaluated 39 AOSD patients (men 64.1%), mean age of 48.8±16.6 years). Out of those, 14 patients (35.9%) were complicated by MAS. These patients showed higher values of ferritin [AOSD: 770.0 (1306.5) ng/mL vs MAS: 2926.3 (4918.5) ng/mL p=0.003] and systemic score (AOSD: 4.6±1.4 vs MAS: 6.9±1.7, pConclusion:Our findings showed a higher prevalence of multiorgan involvement in AOSD patients with MAS, suggesting imaging-based differences, although other studies are needed to fully assess this issue. Pulmonary disease, hepatomegaly, splenomegaly, lymph nodes enlargement, and abdominal effusions were associated with these more aggressive patients.References:[1]Giacomelli R, Ruscitti P, Shoenfeld Y. A comprehensive review on adult onset Still’s disease. J Autoimmun. 2018 Sep;93:24-36.Disclosure of Interests:None declared

Background:Adult onset Still’s disease (AOSD) is a rare systemic autoinflammatory disease and joint involvement is one of its clinical manifestations [1]. Arthritis, either oligoarthritis or bilateral symmetrical rheumatoid arthritis-like polyarthritis, is another common clinical feature of AOSD, with a migrating pattern at the beginning and becoming stable over the time [1].Objectives:The aims of the study were to assess joint involvement in AOSD by using magnetic resonance imaging (MRI), to describe main patterns of involvement, and associated clinical characteristics, and to evaluate the global transcriptomic profile of synovial tissues in AOSD to elucidate possible pathogenic pathways involved with.Methods:AOSD patients, who underwent to magnetic resonance imaging (MRI) exam on joints, were assessed to describe patterns of joint involvement and associated clinical characteristics. Some synovial tissues were collected for RNA-sequencing purposes.Results:In this study, 31 patients with AOSD (mean age 42.3 ± 15.2 years, 54.8% male gender), who underwent to at least one MRI exam on joints, were assessed. The most common MRI finding was the presence of synovitis on 60.5%, mainly in peripheral affected joints. MRI revealed a mild to moderate proliferative synovitis, as thickening of the synovial membrane, with low to intermediate signal intensity on T1-weighted images and intermediate to high signal intensity on T2-fat saturated weighted and STIR images, suggesting the presence of a hyperplastic than of a hypertrophied synovial tissue. Bone oedema and bone erosions were reported on 34.9% and 25.6% MRI exams, respectively. In all patients but one, bone erosions were synchronous with bone oedema, overlapping completely the locations. Assessing clinical characteristics in patients with MRI-erosions, a higher prevalence of splenomegaly, a more frequent chronic disease course, lower levels of erythrocyte sedimentation rate and ferritin was observed.Assessing the synovial tissues of some AOSD patients, a moderate perivascular mononuclear infiltrate in the sub-lining stroma of hip synovial tissues was observed, whereas the lining cells were relatively unremarkable. In addition, interleukin (IL)-1β, IL-6, TNF, and heavy ferritin subunit (FeH) were found on AOSD synovial tissues.An RNA-sequencing analysis assessed the global transcriptomic profile of synovial tissues on AOSD patients and matched-controls. Assessing IL-1 pathway, we found an increased expression of il1a, il1b, il1rap, il1r1, il18r1, and Il18bp on AOSD tissues when compared with controls. In IL-6 pathway, we found an increased expression of il6 and il6st/gp130 on AOSD synovial tissues whereas an increased expression of il6r was shown on the controls. Among genes involved in TNF pathway, tnf, traf1, traf2, tnfaip3 and tnfrsf1a resulted to be more expressed in AOSD synovial tissues than in controls. Finally, fth1 and ftl were more expressed in AOSD patients than controls, when we explored the iron uptake and transport pathway.Conclusion:A peculiar MRI pattern of joint involvement in AOSD was reported; the most common finding was the presence of synovitis, characterised by intermediate to high signal intensity on T2-fat-saturated weighted and STIR images. Bone erosions and bone oedema were also observed. This MRI pattern was associated with a hyper-activation of IL-1, IL-6, and TNF pathways together with a hyper-expression of ferritin genes on AOSD synovial tissues.References:[1]Giacomelli R, Ruscitti P, Shoenfeld Y. A comprehensive review on adult onset Still’s disease. J Autoimmun. 2018;93:24-36.Disclosure of Interests:None declared

Background:The pathogenesis of pSS relies on a complex interplay between both innate and adaptive immune responses in which B cells play a pivotal role. Their chronic aberrant hyperactivation may drive clonal escape and consequent lymphomagenesis [1]. In the last few years, B cells have emerged as potential effector cells, able to release a wide range of cytokines that actively contribute to shape the microenvironment they act in. Recently, IL-40, a novel B cell associated cytokine encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99) was described. Naïve B cells can express IL-40 at both tissue and peripheral level and the stimulation of B cells with IL-4 and TGF-β significantly increases IL-40 release. In addition, human B cells lymphomas are able to constitutively produce IL-40 [2]. Taking into account this emerging evidence and considering the well-known role of IL-4 and TGF-β in pSS pathogenesis, as well as the association with lymphomas, we decided to focus our attention on IL-40 in pSS patients.Objectives:The aim of the present study was to investigate IL-40 expression in the salivary glands of patients affected by pSS and pSS-associated non-Hodgkin’s lymphoma (NHL).Methods:Minor salivary gland biopsies were obtained from 22 patients with pSS and 12 patients with non-specific chronic sialoadenitis (nSCS), included as controls. Paraffine-embedded samples of parotid glands from patients with a previous diagnosis of pSS-associated NHL (n=10) were selected from the biopsy bank of the Pathology Unit of the Ospedale Cervello (Palermo, Italy). Quantitative gene expression analysis by TaqMan real-time PCR and immunohistochemistry (IHC) for IL-40, IL-4, TGF-β1 was performed on salivary glands from patients and controls. The cellular sources of IL-40 among infiltrating inflammatory cells were determined by fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF). Serum IL-40 levels were measured by ELISA in both patients (n=10) and controls (n=9).Results:IL-40 was significantly increased at both protein and mRNA level in the inflamed salivary glands of patients with pSS where a positive strong correlation between the IL-40 mRNA levels and the focus score (FS) was evidenced. The expression of IL-40 in parotid glands of pSS-associated NHL was also markedly increased (Figure 1). IL-40 expression correlated with the presence of IL-4 and TGF-β; both cytokines were significantly increased in pSS at mRNA and protein level. Among infiltrating immune cells, CD19+ B cells resulted the major source of IL-40. However, we identified CD4+, CD8+ T cells and CD68+ macrophages as additional producers of IL-40 in both FACS and IF analysis. The ELISA test also showed a significant increase of serum IL-40 concentration in pSS patients (p value = 0.0190), compared to controls.Conclusion:Our preliminary results suggest that IL-40 may play a role in the pathogenesis of pSS and pSS-associated NHL. To the best of our knowledge, this is the first demonstration of the overexpression of this cytokine in salivary gland tissue and sera in pSS. Moreover, we demonstrated that IL-40 is produced by several cellular types, such as T cells and macrophages, and is not exclusively released by B cells. Further studies are necessary to clarify IL-40 pathways and functions in order to unravel IL-40 possible role in pSS development.References:[1]Mariette X, Criswell LA. Primary Sjogren’s Syndrome. N Engl J Med. 2018;379(1):97.[2]Catalan-Dibene J, Vazquez MI, Luu VP, Nuccio SP, Karimzadeh A, Kastenschmidt JM, et al. Identification of IL-40, a Novel B Cell-Associated Cytokine. J Immunol. 2017;199(9):3326-35.Figure 1.IL-40 overexpression in pSS and pSS-associated lymphoma A, Salivary gland biopsies stained for IL-40 showing a marked increase in IL-40 expression in presence of higher focus score. B, Parotid gland specimens of pSS-associated lymphoma showing intense staining for IL-40.Disclosure of Interests:Chiara Rizzo: None declared, Marianna Lo Pizzo: None declared, Leila Mohammadnezhad: None declared, Vincenzo Luca Lentini: None declared, Diana Di Liberto: None declared, Giulia Grasso: None declared, Piero Ruscitti Consultant of: Pfizer, Novartis, Celgene, Abbvie, Roche, Lilly, Rorberto Giacomelli Consultant of: Pfizer, Novartis, Celgene, Abbvie, Roche, Lilly, francesco ciccia Consultant of: Pfizer, Novartis, Celgene, Abbvie, Roche, Lilly, Giuliana Guggino Consultant of: Pfizer, Novartis, Celgene, Abbvie, Roche, Lilly

Background:During rheumatoid arthritis (RA), a chronic autoimmune disease, the loop existing between inflammation and angiogenesis, characterised by new vessels formation associated with the increased recruitment of inflammatory cells, via the abnormal neo-angiogenesis in the synovial tissues, is considered an early important pathogenic mechanism.Tofacitinib, a potent and selective JAK inhibitor, showed a good profile of safety and efficacy in RA patients, slowing the radiographic progression of the disease. In the last years, many works confirmed that some pro-angiogenic genes are targets of STATs family, and among them, vascular endothelial growth factors (VEGF), a potent pro-angiogenic molecule, may promote the new vessels formation via JAK/STAT pathways.Objectives:The aim of this work was to investigate the inhibiting role of tofacitinib, on the angiogenic mechanisms occurring during experimental model of arthritis.Methods:Healthy control (HC) ECs were stimulated with VEGF and/or tofacitinib and assessed for tube formation and migration, by matrigel and Boyden chamber assay. Furthermore, after ethical approval the experimental model of arthritis was obtained, stimulating 32 mice with collagen (CIA) and 32 mice with PBS (control). At day-19, CIA and controls mice were divided in 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day-35, the mice were scarified and the thickness of paw joints, the synovial vessels and the serum levels of VEGF and Ang-2 were evaluatedResults:In vitro, after tofacitinib-treatment, HC-ECs lose their ability to form vessels and to migrate.In vivo, tofacitinib significantly prevented the increase of paw thickness induced by the collagen administration and reduced the vessel density in synovial tissues of joints, when compared to CIA that did not received tofacitinib. Furthermore, the serum levels of VEGF and Ang-2 were higher in CIA mice, than in control mice. The administration of tofacitinib was able to prevent the VEGF and Ang-2 accumulation in CIA mice.Conclusion:During the last decade, the biological analogies between solid tumors and synovial pannus, and the encouraging results of anti-angiogenic treatments in oncology, lead to increasing interest for angiogenesis as a possible therapeutic target in RA. The present study demonstrated the anti-angiogenic efficacy of tofacitinib, opening a new perspective application for this molecule and improving our therapeutic skill to control the clinical evolution of RA.References:[1]Leblond A et al. Autoimmun Rev 2017;16:594-601.[2]Fleischmann R et al. N Engl J Med 2012;367:495-507.[3]Marrelli A, Autoimmun Rev 2011;10:595-8.Disclosure of Interests:Paola Cipriani Grant/research support from: Actelion, Pfizer, Speakers bureau: Actelion, Pfizer, Paola Di Benedetto Grant/research support from: Paola Di Benedetto received grant from Dompè outside this work., Piero Ruscitti Grant/research support from: Piero Ruscitti received grant from Pfizer outside this work., Speakers bureau: Piero Ruscitti received speaker honoraria BMS, MSD, Ely Lilly, SOBI outside this work, Onorina Berardicurti: None declared, Vasiliki Liakouli Grant/research support from: Vasiliki Liakouli received grant from Pfizer outside this work., Speakers bureau: Vasiliki Liakouli received speaker honoraria from Sanofi Genzyme outside this work., Francesco Carubbi Speakers bureau: Francesco Carubbi received speaker honoraria from Abbvie and Celgene outside this work., Noemi Panzera: None declared, Nicolò Grazia: None declared, Mauro Di Vito Nolfi: None declared, Barbara Di Francesco: None declared, Antonio Maurizi: None declared, Nadia Rucci: None declared, Anna Teti: None declared, Francesca Zazzeroni: None declared, Edoardo Alesse: None declared, Roberto Giacomelli Grant/research support from: Roberto Giacomelli received research grant from Pfizer.This study was supported by an unconditioned Research grant from Pfizer., Speakers bureau: Roberto Giacomelli received speaker honoraria from Abbvie, Roche, Actelion, BMS, MSD, Ely Lilly, SOBI and Pfizer outside this work.

The catastrophic outbreak of acute respiratory distress syndrome induced by SARS-CoV-2 (the coronavirus disease 2019, COVID-19) has strongly confirmed the important role of systemic hyperinflammation, independently of the trigger, as a major cause of death.1 In fact, accumulating evidence suggests that a subgroup of patients with severe COVID-19 is burdened by a cytokine storm syndrome, an overwhelming systemic inflammation with a massive release of pro-inflammatory cytokines. As reported in 150 patients in Wuhan, China, a subset of patients with COVID-19 are characterised by raised levels of ferritin, which identified those patients at higher risk of poor outcome (1297.6 ng/mL in non-survivors compared with 614.0 ng/mL in survivors).1 Although the amplitude of ferritin elevation is not comparable, this finding may resemble what is observed in diseases included under the umbrella of the hyperferritinaemic syndrome.2 This syndrome includes macrophage activation syndrome (MAS), systemic juvenile idiopathic arthritis, adult-onset Still’s disease (AOSD) and catastrophic anti-phospholipid syndrome, which are burdened by severe clinical picture and high mortality rate.2 Ferritin, the common denominator of all these diseases, is an iron storage protein comprising 24 subunits of two types, heavy (FeH) and …

Dear Editor, We read the correspondence by Muravion and Muravion1 about our previous correspondence on a recent clinical trial investigating the efficacy of canakinumab on adult-onset Still’s disease (AOSD)2 3 with interest. In this correspondence, Muravion and Muravion highlighted the role of disease activity score in 28 joints (DAS28) in assessing the disease activity in AOSD, also advocating American College of Rheumatology definitions of clinical response and treat-to-target recommendations.1 This is relevant in the context of rheumatoid arthritis (RA). However, it is well recognised that AOSD is a different disease from RA, considering pathogenic mechanisms, clinical features and therapeutic strategies.4 5 Different from RA, AOSD-associated arthritis, usually an oligoarthritis, is present in two-thirds of these patients, migrating between joints at the very beginning and becoming stable within the course of the disease.6 Although any joint might be affected, wrists, knees and ankles are frequently involved in AOSD arthritis. However, proximal interphalangeal and metacarpophalangeal joints of the hands and small joints of the feet, including the …

We read with great interest the article by Kedor et al 1 on the efficacy of canakinumab, an interleukin (IL)−1β antagonist, on patients with adult-onset Still’s disease (AOSD). In this multicentre, double-blind, randomised, placebo-controlled trial, 36 patients with active joint involvement were enrolled1; this is the largest clinical trial performed on AOSD so far. Moreover, the results of this trial are of considerable interest in this field, considering the challenge of arranging prospective studies on a rare disease. Despite the improvement of many articular secondary measures, the primary outcome, the proportion of patients with a clinically relevant reduction of the articular manifestation measured by change in disease activity score (ΔDAS28(ESR) >1.2) at week 12, did not achieve statistical significance.1 Apparently, this finding seems to be in contrast with the strong scientific rationale, which is behind the study, of inhibiting IL-1β in AOSD and with the confirmed …

Background:The inflammatory contribution to type 2 diabetes (T2D) has suggested new therapeutic targets by using biologic DMARDs designed for rheumatoid arthritis (RA), and IL-1 would be a common pathogenic mediator, suggesting a possible common therapeutic target [1]. In TRACK study, a multicentre open-label, randomised controlled trial, anakinra, a human interleukin-1-receptor antagonist, showed to induce a significant improvement of metabolic alteration whereas TNFi did not show any significant improvement on that, after both 3 months and 6 months of therapy (crude difference of 0.93 of glycated haemoglobin, HbA1c% between groups) [2]. Concerning RA, a progressive reduction of disease activity was observed in both groups [2].Objectives:Since TRACK study has been prematurely stopped for “early benefit” after 6 months of follow-up, in this work, we aimed at investigating how long lasted the improvement of HbA1c% and of RA disease activity, considering the original scheduled 24 months follow-up. We also assessed the rate of antidiabetic and anti-rheumatic therapies reduction and stoppage.Methods:This study was designed as a multicentre, open-label, randomised controlled trial, enrolled participants, with RA and T2D, in 12 Italian Rheumatologic Units, between 2013 to 2016. Participants were randomised to anakinra or to a TNFi and the primary endpoint was the change in HbA1c% (EudraCT: 2012-005370-62; ClinicalTrial.gov:NCT02236481). In this further evaluation, we assessed how long lasted the improvement of HbA1c% and of RA disease activity, considering the original scheduled 24 months follow-up, and the rate of antidiabetic and anti-rheumatic drugs, mainly focusing on steroids, reduction and stoppage.Results:In TRACK study, 39 participants with RA and T2D (age 62.72 ± 9.97, 74.4% female gender) were randomised to anakinra or to TNFi; the majority of participants had seropositive RA disease (rheumatoid factor and/or ACPA 70.2%) with active disease (DAS28: 5.54 ± 1.03; C-reactive protein 11.84 ±9.67 mg/L, respectively) and all participants had T2D (HbA1c%: 7.77 ±0.70, fasting plasma glucose: 139.13 ±42.17 mg). Considering the last available observation, a maintenance of reduced levels of HbA1c% was observed in anakinra-treated participants (Baseline: 7.73% ±0.67; 6 months: 6.70% ±0.67; last follow-up: 6.60% ±0.52). Paralleling with HbA1c%, a significant reduction of dosages of antidiabetic therapies was observed in anakinra-treated patients, with a percentage of patients who discontinued any anti-diabetic therapy. Conversely, an intensification of antidiabetic therapies was reported in TNFi-treated participants. Concerning RA, the clinical response was maintained during the whole follow-up, although a larger percentage of anakinra-treated participants discontinued the concomitant steroids therapy.Conclusion:In this study, we observed the benefit of IL-1 inhibition in patients with RA and T2D, reaching the therapeutic targets of both diseases, which lasted longer than first 6 months of follow-up. Although the limitations due to open-label design and the necessity of further confirmatory studies, our results could suggest the concept that IL-1 inhibition may be considered a targeted therapeutic strategy for RA and T2D.References:[1]Donath MY. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat Rev Drug Discov. 2014;13: 465-476.[2]Ruscitti P et al. Anti-interleukin-1 treatment in patients with rheumatoid arthritis and type 2 diabetes (TRACK): A multicentre, open-label, randomised controlled trial. PLoS Med 16(9): e1002901.Disclosure of Interests:Piero Ruscitti Grant/research support from: Pfizer, Speakers bureau: BMS, MSD, Ely Lilly, SOBI, Saverio Alvaro: None declared, Vasiliki Liakouli Grant/research support from: Pfizer, Speakers bureau: Sanofi Genzyme, Federica Sensini: None declared, Paola Cipriani Grant/research support from: Actelion, Pfizer, Speakers bureau: Actelion, Pfizer, Roberto Giacomelli Grant/research support from: Actelion, Pfizer, Speakers bureau: Abbvie, Roche, Actelion, BMS, MSD, Ely Lilly, SOBI, Pfizer

Background:The concept of ‘hyperferritinemic syndrome’ has recently been proposed, suggesting high levels of ferritin as pathogenic pro-inflammatory mediator [1] Ferritin is an intracellular iron storage protein, comprising 24 subunits, heavy (H) and light (L) based on molecular weight. The H-/L subunits ratio may be different in tissues, since the ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin) may be observed in different tissues, according to pathophysiologic status [1].Objectives:We aimed to assess the presence of H- and L-ferritin as well as of CD68/H-ferritin and CD68/L-ferritin in bone marrow (BM) biopsies of adult macrophage activation syndrome (MAS) patients. In the same patients, we matched the findings of BM biopsies with sera to identify the main represented subunits of ferritin. Furthermore, we evaluated effects of ferritin, L-ferritin, and H-ferritin on human monocytes, assessing pro- and anti-inflammatory cytokines, and expression of NLRP3 inflammasome. Finally, we checked the ability of monocytes, which were treated with ferritin, to stimulate or not the proliferation of peripheral blood mononuclear cells (PBMCs).Methods:Immunofluorescence analysis was performed to investigate the tissue presence of L- and H-ferritin in BM biopsies as well as of CD68/H-ferritin and CD68/L-ferritin. Liquid chromatography mass spectrometry (LC-MS/MS) based proteomics was performed to identify L- and H-ferritin in sera proteins. Human monocytes were cultured with M-CSF for 7 days and, after that, treated with ferritin, H-ferritin, and L-ferritin at 10nM, for 120 and 240 minutes. After stimulation, IL-1β, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF, and VEGF were assessed by RT-PCR and, in case of positive finding, evaluated by western blot. NLRP3 inflammasome was also assessed. Finally, the proliferation of PBMCs when co-cultured with ferritin-treated monocytes was tested by a specific proliferation assay.Results:Immunofluorescence showed an increased H-ferritin expression in the BMs of MAS patients, whereas L-ferritin did not. Conversely, LC-MS/MS identified that the L-ferritin was the dominant form, after stringent probability matching.In vitro, H-Ferritin induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Ferritin also induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Effects on pro-inflammatory cytokines were more marked with H-ferritin than ferritin. Conversely, no significant effects were retrieved analysing IFN-γ, IL-10, TGF-β, and VEGF after 240 minutes, after ferritin and H-ferritin stimulation. Furthermore, both ferritin and H-ferritin induced a direct effect on NLRP3 inflammasome. Finally, monocytes, which were treated with H-ferritin, stimulated the proliferation of co-cultured PBMCs.Conclusion:In our work, results showed the presence of H-ferritin and CD68/H-ferritin cells in BM biopsies of MAS patients, by immunofluorescence. Conversely, LC-MS/MS identified L-ferritin in sera proteins of those patients. Furthermore, pro-inflammatory effects of ferritin and, particularly, of H-ferritin on human monocytes were observedin vitro, increasing pro-inflammatory cytokines and NLRP3 inflammosome. Finally, H-ferritin-treated monocytes stimulated the proliferation of co-cultured PBMCs.References:[1]Rosario C, et al. BMC Med 2013; 11:185.Disclosure of Interests:Piero Ruscitti Grant/research support from: Pfizer, Speakers bureau: BMS, MSD, Ely Lilly, SOBI, Paola Di Benedetto Grant/research support from: Paola Di Benedetto received grant from Dompè outside this work., Onorina Berardicurti: None declared, Noemi Panzera: None declared, Federica Sensini: None declared, Paola Cipriani Grant/research support from: Actelion, Pfizer, Speakers bureau: Actelion, Pfizer, Roberto Giacomelli Grant/research support from: Actelion, Pfizer, Speakers bureau: Abbvie, Roche, Actelion, BMS, MSD, Ely Lilly, SOBI, Pfizer

Background Macrophage activation syndrome (MAS) is a rare, life-threatening disease in which early diagnosis and aggressive therapeutic strategy may improve the outcome [1]. Due to its rarity, epidemiologic data are still lacking. Hyperferritinemia is frequently associated with MAS and might modulate the cytokines storm the latter contributing to the development of multi-organ failure [2]. Objectives In the current study, we aimed to investigate clinical data, treatments, and outcome of adult MAS patients secondary to autoimmune rheumatic disease. Methods We retrospectively investigated clinical data, treatments, and outcome of 40 adult MAS patients secondary to autoimmune rheumatic disease. For all the MAS patients, the following clinical characteristics were recorded: age, gender, values of serum ferritin, erythsedimentation rate (ESR) and C-reactive protein (CRP), treatments, concomitant comorbidities, time of observation and outcome. Furthermore, we evaluated the correlations among these clinical features and MAS-associated death. Results Forty consecutive MAS patients, 15 men (37.5%) and 25 women (62.5%), whose mean age at diagnosis was 48.20±14.10 (mean±SD) were enrolled. Thirty out of 40 enrolled patients reported an history of adult onset Still9s disease (AOSD) (75%), 8 patients an history of systemic lupus erythematosus (20%), 1 patient an history of systemic sclerosis (2.5%), and 1 an history of ankilosing spondylitis (2.5%). Twenty-four out of enrolled patients (60%) did not show any comorbidity, and MAS was diagnosed during an AOSD flare. A severe infections was associated with MAS in the other patients. The laboratory data (mean±SD) reported a strong increase of inflammatory markers: i. serum ferritin 6842.70±7569.70 ng/mL; ii. ESR 63.87±30.19 mm/hr; iii. CRP 51.59±46.92. All the patients were treated with pulsed high dosage of methylprednisolone (1000 mg/daily for a maximum of 6 cycles) and 16 patients (16, 40%) received a combination therapy with: cyclosporin (13, 32.5%); methotrexate (6, 15%); intravenous immunoglobulin therapy (3, 7.5%); anakinra (3, 7.5%); tocilizumab (1, 2.5%). MAS-associated death occurred in 17 patients (42.5%). By using Cox regression proportional hazard model of survival of enrolled MAS patients, we observed that: older age [p=0.017, EXP(B): 1.305, 95%CI: 1.006–1.365], increased serum ferritin levels [p=0.001, EXP(B): 1.776, 95%CI: 1.030–2.728], increased CRP levels [p=0.029, EXP(B): 1.511, 95CI%: 1.021–1.651], higher number of comorbidities [p=0.01, EXP(B): 1.751, 95%CI: 1.142–2.2686], and the use biologics [p=0.015, EXP(B): 2.915, 95%CI: 1.233–6892] were significantly associated with MAS-associated death. Conclusions Adult MAS is still associated with higher mortality rate. Some clinical features at diagnosis, such as older age, increased serum ferritin and CRP levels and higher number of comorbiditis, may be associated with MAS-associated death. References Ramos-Casals M et al, Adult haemophagocytic syndrome. Lancet 2014; 383: 1503–16. Rosario C et al, The hyperferritinemic syndrome: macrophage activation syndrome, Still9s disease, septic shock and catastrophic antiphospholipid syndrome, BMC Med 11, 2013,185. Disclosure of Interest None declared

Background Systemic Sclerosis (SSc)-heart disease (SSc-HD), although often clinically silent, significantly reduces the life-expectancy in these patients [1]. The cardiac magnetic resonance (CMR) is recognized useful tool for the diagnosis of SSc-HD and cardiac stress tests might be a helpful technique to highlight the occult myocardial involvement, which cannot be detected at rest due to compensatory mechanisms [2,3]. Objectives To evaluate the possible occult cardiac involvement in asymptomatic SSc patients by pharmacological stress, rest perfusion and delayed enhancement CMR, to identify as earlier as possible those patients at higher risk of cardiac related mortality and needing specific cardiovascular treatments. Methods Sixteen consecutive patients with definite SSc fulfilling the ACR/EULAR 2013 classificative criteria [4] in less than 1 year from the onset of Raynaud9s phenomenon underwent pharmacological stress, rest perfusion and delayed enhancement CMR. No enrolled patient showed signs and/or symptoms suggestive for cardiac involvement, confirmed by both the 12-lead ECG examination and echocardiography. No patient showed traditional cardiovascular risk factors. Patients, in which CMR technique showed occult cardiac involvement, underwent to heart computed tomography (CT) scan to evaluate a possible coronary disease. Results Stress perfusion defects, in the left ventricle, were detected in 6 out of 16 (37.5%) patients. A sub-endocardial defects and/or a ventricular mid-myocardial layer stress perfusion defects were observed. Our results confirmed, in these 6 patients, a normal flow distribution of the explored coronary arteries by CT scan. The presence of CMR stress perfusion defects did not correlate with any clinical feature of patients. Conclusions Myocardial stress perfusion defects may be early detected, by pharmacological stress perfusion CMR, a reliable and sensitive technique for the non-invasive evaluation of SSc-HD, in patients with SSc of recent onset. These defects seems to be independent from traditional risk factors and associated comorbidities, suggesting that are a specific hallmark of the disease. An early detection of myocardial involvement might suggest starting of vasodilatative therapies in these patients. References Allanore Y, et al. Primary myocardial involvement in systemic sclerosis: evidence for a microvascular origin. Clin Exp Rheumatol 2010;28:S48–53. Di Cesare E, et al. Early assessment of sub-clinical cardiac involvement in systemic sclerosis (SSc) using delayed enhancement cardiac magnetic resonance (CE-MRI). Eur J Radiol. 2013;82:e268–73. Hachulla AL, et al. Cardiac magnetic resonance imaging in systemic sclerosis: a cross-sectional observational study of 52 patients. Ann Rheum Dis. 2009;68:1878–84. van den Hoogen F, et al. 2013 Classification Criteria for Systemic Sclerosis An American College of Rheumatology/European League Against Rheumatism Collaborative Initiative. Arthritis Rheum. 2013;65:2737–47. Disclosure of Interest None declared

Background GO-MORE Trial investigated the use of Golimumab (GLM) in rheumatoid arthritis (RA) in 3280 patients worldwide [1]. At present, the burden of arthritis is greater in poorer countries than in developed countries due to socioeconomic disparities [2], thus suggesting the usefulness of subgroup investigations. Objectives We evaluated the GLM as add-on therapy for RA patients in the Italian cohort of GO-MORE trial and we compared the baseline characteristics and efficacy after 6 months between Italian patients and the enrolled patients worldwide. A limit of these analyses is that they are post hoc in nature and many tests were performed without adjustments for multiplicity. Methods Ninety-eight Italian patients with active RA, fulfilling the 1987 ACR criteria, naive for biologic drugs were enrolled. Statistical analyses were performed to assess: i. the differences in baseline characteristics; ii. the efficacy after 6 months; between Italian and Rest of the World GO-MORE populations. Results The majority of Italian patients were older than the worldwide patients (p=0.01). Compared to Rest of the World population, Italian patients showed a lower value of DAS28-ESR (p=0.045), DAS28-CRP (p=0.0001), and a significantly short disease duration (≤2 years) (p=0.045). Differently from the Rest of the World patients, the large majority of Italian patients received biologic therapy after the failure of the first sDMARD (p Conclusions The analysis of the Italian GO-MORE subset confirms that differences among patients may be shown, depending from different approaches in different health systems. The Italian cohort (early onset of the disease and already fulfilling the 1987 ACR criteria) is probably affected by a more severe disease with poor outcome [4], but the efficacy in this subset response rate of Italian subset did not differ from worldwide patients. GLM in the Italian patients showed a favourable benefit/risk profile and the positive autoinjection experience may help for patient9s compliance and survival of the treatment. References Combe B, et al. Efficacy and safety of golimumab as add-on therapy to disease-modifying antirheumatic drugs: results of the GO-MORE study. Ann Rheum Dis. 2014;73:1477–86. Sokka T, et al. QUEST-RA: quantitative clinical assessment of patients with rheumatoid arthritis seen in standard rheumatology care in 15 countries. Ann Rheum Dis. 2007;66:1491–6. Schulze-Koops H, et al.Factors influencing the patient evaluation of injection experience with the SmartJect autoinjector in rheumatoid arthritis. Clin Exp Rheumatol. 2015;33:201–8. Bykerk VP, et al. The Canadian Early Arthritis Cohort (CATCH): patients with new-onset synovitis meeting the 2010 ACR/EULAR classification criteria but not the 1987 ACR classification criteria present with less severe disease activity. J Rheumatol. 2012;39:2071–80. Disclosure of Interest R. Giacomelli Grant/research support from: RG has received a grant from MSD for educational purpose, P. Ruscitti: None declared, V. Liakouli: None declared, P. Cipriani: None declared, M. Mecchia Employee of: MM is an employee of MSD Italy

Background Adult onset Still9s disease (AOSD) is an inflammatory disease, characterized by high spiking fevers, arthritis, salmon-pink erythema and multivisceral involvement [1]. During AOSD, exceptionally high serum levels of ferritin may be observed and they might contribute to production of proinflammatory molecules [2]. Ferritin is composed by 24 subunits, heavy (H) subunits and light (L) subunits. The ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin) may be recognized in different tissues [3]. Objectives To investigate the skin tissue expression of both H-and L-ferritin and the number of macrophages expressing these molecules, in the inflammatory cutaneous infiltrate from 10 AOSD patients with persistent (more than 24–36 hours) cutaneous lesions. In addition, we performed statistical analysis to evaluate whether these results might correlate with the severity of the disease. Methods We obtained skin samples from 10 consecutive patients with active AOSD with persistent cutaneous lesions and 10 healthy controls (HC). An immunofluorescence analysis was performed evaluating the tissue expression of both H- and L-ferritin and the number of macrophages expressing these moleculkes. Serum levels of ferritin, ESR, CRP, Pouchot9s score were also recorded. Results We observed an increased H-ferritin expression in the skin samples of AOSD patients, when compared with HC (p Conclusions Our data show an imbalance between the production of L- and H-ferritin in the skin of AOSD patients, associated with a strong infiltrate of CD68+/H-ferritin+ cells in the same samples suggesting a possible pathogenic role. Furthermore, a correlation between the levels of H-ferritin in the skin of these patients and the disease activity was observed. References Gerfaud-Valentin M, et al. (2014) Adult-onset Still9s disease. Autoimmun Rev 13:708–22. Rosario C, et al (2013) The hyperferritinemic syndrome: macrophage activation syndrome, Still9s disease, septic shock and catastrophic antiphospholipid syndrome. BMC Med 11:185. Recalcati S, et al (2008) New functions for an iron storage protein: the role of ferritin in immunity and autoimmunity. J Autoimmun 30:84–9. Disclosure of Interest None declared

Background Adult onset Still9s disease (AOSD) is rare inflammatory disease [1]. Three different patterns of AOSD have been identified: i. monocyclic pattern, characterized by a systemic single episode; ii. polycyclic pattern, associated with multiple flares, separated by remissions; iii. chronic pattern, related to a persistently active disease with associated polyarthritis [2]. Until now, the treatment of AOSD remains largely empirical, lacking controlled clinical trials [1]. Objectives We aimed to investigate clinical data of AOSD patients and any possible correlation among these features and the outcome of patients. Methods Clinical data of 100 AOSD patients were recorded. All patients fulfilled the Yamaguchi AOSD diagnostic [3]. Gender, age, clinical features, disease activity Pouchot9s score [4], complications, serum ferritin, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), treatments, comorbidities, patterns and AOSD-associated death, were reported. Furthermore, we evaluated any possible correlation among these clinical features and the outcome of patients, by using linear regression analysis. Results One hundred consecutive AOSD patients (66 men, 34 women), whose age at diagnosis was 45.35±16.23 (mean±SD), were enrolled. All patients experienced fever, 86 patients showed joints involvement, 79 patients showed splenomegaly, skin rash was present in 78 patients, 62 patients showed hepatic involvement, 57 patients showed enlargement of lymph nodes. The Pouchot9s score, 6.11±2.02 (mean±SD), confirmed the activity of the disease. Sixteen patients experienced different complications, mainly the macrophage activation syndrome. A strong increase of inflammatory markers (mean±SD) was observed: serum ferritin was 2560.07±3726.64 ng/mL, ESR was 67.28±26.65 mm/hr, CRP was 78.35±72.76. All the patients received steroids at different dosages, and 55 patients were treated with steroids and immunosuppressive drugs, mainly methotrexate and ciclosporin. Thirty-two patients were treated with biologic agents: 5 with anakinra, 8 with tocilizumab, and 19 with TNF inhibitors. Twenty-nine patients showed the monocyclic pattern, 22 patients showed the polycyclic pattern, 33 patients showed the chronic pattern. AOSD-associated death occurred in 16 patients. Regression analysis showed that Pouchot9s score (p=0.011), the presence of any complication (p Conclusions This study provides information on the clinical, laboratory, therapeutic, and prognostic features in a large cohort of AOSD patients. The Pouchot9s score, the presence of complications and comorbidities were significantly associated with the AOSD-associated death. References Gerfaud-Valentin M, et al. Adult-onset Still9s disease, Autoimmun Rev 2014;13,708–722. Cush JJ, et al. Adult-onset Still9s disease. Clinical course and outcome. Arthritis Rheum 1987;30:186–9425. Yamaguchi M, et al. Preliminary criteria for classification of adult Still9s disease. J Rheumatol 1992;19:424–30. Pouchot J, et al. Adult Still9s disease: manifestations, disease course, and outcome in 62 patients. Medicine (Baltimore) 1991;70:118–36. Disclosure of Interest None declared

Background A better understanding about the mechanisms involved in the pathogenesis of type 2 diabetes mellitus (T2DM) showed that inflammatory cytokines such as tumor TNF-α and IL-1β play a pivotal role, mirroring data largely reported in rheumatoid arthritis (RA) [1]. Both preclinical and clinical studies confirmed the usefulness of IL-1β antagonism therapy in both diseases [2]. IL-1β is mainly produced by monocytes/macrophages. Hyperglycaemia may be able to modulate, in the cytoplasm of these cells, the assembly of a cytosolic multiprotein platforms, composed by 3 molecules: NLRP3 sensor protein; ASC adaptor protein; caspase-1, the so called NLRP3-inflammasome, where, the inactive pro-IL-1β is cleaved into active form, via caspase-1 activity [3]. Objectives We evaluated the production of IL-1 β and TNF-α, in peripheral blood monocytes (MO) of patients affected by RA or T2DM or both diseases, in order to understand if, an alteration of the glucose metabolism may influence their pro-inflammatory status. Methods We enrolled 10 RA patients, 10 T2DM patients and 10 patients affected by both the diseases (T2DM/RA patients), and 5 healthy controls (HC). From these patients, MO were isolated from human peripheral blood mononuclear cells using CD14 selection. After that MO were incubated with 11 mmol/1 glucose (11G) and 33 mmol/1 glucose (33G), for 24 hours, and we evaluated by ELISA the concentration of both IL-1β and TNF-α released in cultured MO supernatants, the relative mRNA expression by qRT-PCR and the western blot analysis of NLRP3. Due to the non parametric distribution of our data the Mann-Whitney U test was used as appropriate for analyses. Statistical significance was expressed by a p value Results Our data showed, after 24 hours of incubation with 11G, a significantly increase of IL-1β in supernatants of T2DM, RA, and T2DM/RA cultured MO, when compared with HC (p Conclusions Our study showed an increased production of IL-1β by MO obtained from patients affected by both RA and T2DM via NLRP3-inflammasome activation suggesting a potential IL-1β target therapy. References Donath MY, et al. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol. 2011;11:98-107. Eguchi K, et al. Macrophages and islet inflammation in type 2 diabetes. Diabetes Obes Metab. 2013;3:152-158. Schroder K, et al. The NLRP3 inflammasome: a sensor for metabolic danger? Science. 2010;327:296-300. Disclosure of Interest None declared

Background Systemic Sclerosis (SSc) is characterized by vascular alteration with a progressive loss of capillaries resulting in insufficient blood flow and chronic peripheral hypoxia, associated to a failure of reparative angiogenesis, followed by progressive fibrosis of skin and internal organs[1]. An impaired production of angiogenic molecules is involved in this dysfunctional angiogenesis [2]. Tie2 is a trans-membrane receptor, exclusively expressed on the surface of endothelial cell (EC). Tie2 modulates the vessels quiescence, or alternatively, induces angiogenesis, via the interaction with its ligands, Ang1 and Ang2. Of note, activation of endothelial Tie2 signalling, by Ang1, which is produces by perivascular cells, enhances ECs barrier integrity and endothelial-pericyte interaction. On the contrary, Ang2, produced by ECs, normally acts as an Ang1 antagonist [3]. Objectives The aim of the present study was to assess if an impaired cross-talk between ECs and perivascular mesenchymal stem cells (MSCs), during SSc, may affect the normal interaction among Ang1, Ang2 and Tie2 thus contributing to the impaired angiogenesis. In this work, bone marrow derived MSCs were used as pericytes surrogate, considering that, perivascular cells share surface markers and differentiative ability with bone marrow MSCs, and MSCs express pericyte markers and cooperate with endothelial cells to form a vascular network, supporting the concept that pericytes are members of the adult multipotent MSCs family [4]. Methods We investigated Ang1, Ang2 and their receptor performing co-cultures with ECs and bone marrow MSCs obtained from patients and healthy controls (HC). After 48 hours, cells were sorted and analysed for molecular assays. Furthermore, we investigated, by ELISA assay, the proteins released in the supernatants. Finally, we silenced Ang-1 expression in HC-MSCs by Ang1-siRNA. Results At molecular level, SSc-MSCs, cultured alone, expressed lower amount of Ang1 when compared to HC-MSCs. After co-culture, a significant decreased of Ang1 mRNA levels was observed in the SSc-MSCs/SSc-ECs. On the contrary, SSc-ECs expressed higher levels of Ang2 and Tie2 in each co-culture condition, when compared to the expressions of cells cultured alone. The WB and ELISA assays mirrored the results observed in gene expression. HC-MSCs transfected with Ang1-siRNA lacked the ability to support the formation of tube like structure. Conclusions In this work we provided evidence that an imbalance of Ang1/Ang2 molecules and a decreased expression of their receptor, Tie2, during ECs-perivascular MSCs interplay, may modulate vessel stability, and vascular tube formation, thus contributing to the angiogenic alteration observed during SSc. References Cipriani P et al. Autoimmun Rev 2011,10:641-646. Cipriani P et al. Arthritis Res Ther 2013,16:442. Fukuhara S et al. Histol Histopathol 2010,25:387–396. Cipriani P et a. Angiogenesis 2013,16:595-607. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is an autoimmune disorder characterized by microangiopathy and immunological abnormalities evolving into fibrosis. Endothelial damage occurs early in the disease. EGFL7, is a recently identified molecule expresses in vascular endothelial cells (ECs) and their mesodermal progenitors. EGFL7 controls blood vessel development by promoting ECs migration, proliferation, sprouting, adhesion (1,2). Its alteration results in abnormal vessel formation. Objectives We hypothesized that EGFL7 might play an important role in regulating SSc angiogenesis/vasculogenesis. Methods EGFL7 expression was investigated in the involved skin of 33 SSc patients, according to the different subsets (16 diffuse and 17 limited SSc) and stages (16 early and 17 late) of the disease and 27 healthy controls (HC), by using immunohistochemistry, immunofluorescence, RT-PCR analyses and Western Blot (WB). Furthermore, placenta biopsies from 4 healthy uncomplicated pregnancies were used as physiological angiogenesis controls. Microvascular endothelial cells (MVECs) and fibroblasts (FBs) both from patients and HC were also analyzed for EGFL7, by confocal laser scanning microscopy, RT-PCR analysis and WB. Results EGFL7 expression is clearly up-regulated in the placenta, that represents the physiological model of angiogenesis as observed by immunohistochemistry, immunofluorescence, RT-PCR and WB analyses, when compared to skin of HC and patients. Moreover, patients in the early stage and diffuse form display lesser expression of EGFL7 when compared to patients with limited subset, late stage, and HC, respectively. Cultured SSc-MVECs express a significantly higher amounts of EGFL7 mRNA in the early stage and lowest expression in the later stages of the disease, not mirroring the skin findings and suggesting a post transcriptional modification. Furthermore, cultured SSc-FBs express lower mRNA and protein levels of EGFL7. Interestingly, in cultured SSc and HC FBs only SSc myofibroblasts (a-SMA +) showed a strong immunopositivity for EGFL7, suggesting a role of this factor in the EndMT (endothelial-mesenchymal transition) transition. Conclusions We report the first observation of an abnormal expression of EGFL7 in the skin and in the myofibroblasts of the SSc patients, compared to controls which is generally considered a specific endothelial molecule. The alteration of EGFL7, seems to be involved in defective angiogenesis of SSc and in the modulation of EndMT, that leads to the fibroproliferative vasculopathy. References Campagnolo L et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol. 2005 Jul;167(1):275-84 Schmidt M, et al. EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development 2007 Aug;134(16):2913-23. Epub 2007 Jul 11. Disclosure of Interest None Declared

Background BM-MSCs are currently being studied to determine their potential role in the pathogenesis of autoimmune diseases. Experimental evidence have shown that they exhibit an anti-proliferative effect on T lymphocytes. In this setting BM-MSCs show a specific immunomodulatory function in inducing regulatory phenotype and function in T cells. T regulatory lymphocytes are a population of CD4+ CD25high Foxp3+ cells which play an important role in the induction of peripheral tolerance. SSc is a complex autoimmune disease, characterized by fibrosis of the skin and various internal organs. Our previous findings suggesting that BM-MSCs derived from SSc show a premature senescence, probably related to several pathologic stimuli which these cells encountered during their lifetimes. Objectives The aim of this work was to demonstrate that BM-MSCs are early senescent, assessing the activity of beta-galactosidase and cell cycle inhibitors (p53 and its downstream gene, p21) and investigate whether during SSc, BM-MSCs, although senescent, maintain their immunosuppressive properties, when co-cultured with T lymphocytes. Methods After ethical commitment approval MSCs were isolated from BM obtained from 10 SSc and 10 healthy controls (HC). At third passage, the cell cycle was analyzed by flow cytometry. The beta-galactosidase activity was measured by counting positive cells after staining with X-Gal. mRNA and proteins of p21 and p53 were measured by western blot and qRT-PCR. The treatment of the cells for 24h with doxorubicin was used to induce senescence. The immunomodulatory property of BM-MSCs was tested by co-culturing these cells with CD4+ T cells. Results Both HC and SSc BM-MSC showed stromal phenotype, and normal multidifferentiative ability. The study of cell cycle showed that about 80% of cells were in G0-G1 phase, without significant differences between SSc and HC. Interestingly, the SSc-MSCs showed an increase in beta-galactosidase compared to HC-MSC. After treatment with doxorubicin, the percentage of beta-galactosidase positive stained cells in SSc were higher with respect to HC, thus outlining that SSc cells show greater sensitivity to drug treatment. As far as the expression of p21 was concerned, intriguingly, we found a significant increase in SSc-MSCs compared with HC-MSCs. Doxorubicin treatment abolished p21 activation and, on the other hand, elicited p53 induction both in SSc and HC BM-MSCs. This regulation of p21/p53 machinery could be an attempt of BM-MSCs to bypass the doxorubicin induced. This protective mechanism seems lesser active in SSc cells. The above results were further confirmed by qRT-PCR. We found that senescent SSc MSC showed a preserved immunomodulatory activity maintaining the immunosuppressive effect and the ability to induce a regulatory phenotype on T as HC BM-MSCs. Conclusions SSc BM-MSC showed an increase of senescence biomarkers, suggesting premature aging, probably related to several pathologic stimuli. Although the early aging of SSc BM-MSCs, the finding that these cells showed a normal immunoregulatory activity, suggesting their potential to recover by activating a sort of adaptive mechanism. References Cipriani P et al. AR56(6). Landgraf K et al. Transpl Immunol 2011 Sep;25. Disclosure of Interest None Declared

Background B lymphocytes are key players in the development of chronic autoimmune sialoadenitis and ectopic lymphoneogenesis is a milestone in the natural history of Sjogren’s syndrome (SS) (1). Much effort has been spent to shed some light on the underlying mechanisms and to date several pathways have been identified. Recruitment and activation of B cells in target organs are orchestrated by a specific milieu of well characterized cytokines and chemokines (2). In this setting, targeted therapies aimed to delete autoreactive B cell clones or B-cell activating factors have been proposed. Recently, growing evidence about the positive effects of anti-CD20 monoclonal antibody rituximab (RTX) in SS pointed out a possible therapeutic application in these patients. However conclusive data about the effective role of RTX in glandular microenvironment are still lacking. Objectives Aim of the present study was to characterize the different histopathological and molecular features of salivary gland infiltrate following either conventional therapies or RTX in SS. Methods Eight females with SS underwent minor salivary gland (MSG) biopsy at the diagnosis and following either conventional therapies or RTX. Four females with sicca symptomatology but neither histologic nor sierologic features of SS acted as controls. Serial sections of 3μm thickness were cut and stained with hematoxylin and eosin for the assessment of several histologic scores according to literature (3). Immunofluoscence staining for anti-CD3, anti-CD20, anti-CD21 was performed to identify the grade of lymphoid organization and the presence of germinal center-like structures. Sections were counterstained with DAPI and analyzed with Olympus BX53 microscope. Part of MSG were processed for real time PCR and the following molecules, involved in inflammation and lymphoid organization, were tested: LTa, LTb, IL-1b, TNFa, BAFF, AID, CXCR4, CXCL12, CXCR5, CXCL13, CCR7, CCL19, CCL21. Results We observed that in SS-MSG the levels of proinflammatory cytokines and chemokines were increased with respect to control (all p Conclusions Our results confirm that RTX is currently the only therapeutic agent able to interfere with the perpetuation of salivary gland damage during SS. Therefore, selective targeting of pathogenic B cells appears to be a promising approach to restore normal glandular function. References Voulgarelis M et al, Nat Rev Rheumatol 2010;6:529-537. Youinou P et al, Arthritis Rheum 2010;62 (9):2605-2610. Christodoulou MI et al, Journal of Autoimmunity 2010;34:400-407. Disclosure of Interest None Declared

Background MVECs damage is a central event in the pathogenesis of SSc leading to capillaries rarefaction and consequent ischemia. VEGF is the best characterized angiogenic growth factor; its increase after ischemia is a timed controlled event, acting only in the first stages of new blood vessels formation when it regulates ECs sprouting and proliferation. During SSc, VEGF lacks this tight control and is strongly up-regulated over time both at local and systemic levels. Caveolae, specialized plasma membrane microdomains, abundant in ECs, and caveolin-1 (cav-1), their principal residual protein, compartmentalize VEGF-induced signaling toward angiogenesis. Objectives Here we aimed to prove whether caveolae/cav-1 act as platform for organizing and compartmentalizing VEGF/VEGFR2 signaling in MVECs during SSc. In fact an abnormal cav-1 expression in microvascular endothelium, may contribute to angiogenic defective response during the disease. Methods After ethical approval skin was collected from 10 patients with SSc and used for immunofluorescence (IF), qPCR and MVECs isolation. Normal skin was obtained from healthy donors (HC) who underwent surgery for trauma. Cav-1 detection and its co-localization with VEGFR-2 was perfomed on skin sections and MVECS isolated from SSc patients and compared to HC by IF, immunoprecipitation and Western Blot (WB). qPCR analysis was used to assess mRNA cav-1 expression level both in SSc skin and MVECs. To determine whether VEGF treatment could affect cav-1/VEGFR2 colocalization in MVECs during SSC, all the above was assessed after culturing these cells in the presence of VEGF. Results We found that VEGFR-2 colocalizes with cav-1 in blood vessels within SSc skin, as already observed in HC skin. However semi-quantitative analysis of IF cav-1 expression level revealed lower expression in SSc MVECS with respect to HC skin. This result was confirmed by qPCR, showing cav-1 was down-regulated in MVECs isolated from SSc patients when compared to HC cells. When we assessed cav-1/VEGFR.2 co-localization within MVECS, we observed that VEGFR-2 was localized in the caveolae directly bound by cav-1. This observation was further confirmed by cav-1 immunoprecipitation and WB for VEGFR-2 in SSc MVECs, without differences from HC. Interestingly, after culturing these cells in the presence of VEGF, we found a disruption of cav-1/VEGFR-2 co-localization both in HC and SSc MVECs, without cav-1 mRNA levels re-modulation Conclusions During SSc cav-1 expression down-regulation within MVECs, could possibly explain the dysfunctional, ineffective VEGF angiogenic signaling. This mechanism might maintain the redundancy of deregulated VEGF abundance in a vicious loop, strongly contributing to not compensative angiogenic response during SSc. References Del Galdo Fet al Curr Opin Rheumatol. 2008 Nov;20(6). Feng L et al J Cell Physiol. 2011 Aug 9 [Epub ahead of print]. Liao WX et al Mol Endocrinol. 2009 Sep;23(9). Disclosure of Interest None Declared