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Active vitamin D [1,25(OH)2D3—calcitriol] is a secosteroid hormone whose receptor is expressed on all cells of the immune system. Vitamin D has a global anti-inflammatory effect and its role in the management of a SARS-CoV-2 infection has been investigated since the beginning of the COVID-19 pandemic. In this narrative review, the laboratory and clinical results of a vitamin D supplementation have been collected from both open-label and blinded randomized clinical trials. The results are generally in favor of the utility of maintaining the serum concentrations of calcifediol [25(OH)D3] at around 40 ng/mL and of the absolute usefulness of its supplementation in subjects with deficient serum levels. However, two very recent large-scale studies (one open-label, one placebo-controlled) have called into question the contribution of vitamin D to clinical practice in the era of COVID-19 vaccinations. The precise role of a vitamin D supplementation in the anti-COVID-19 armamentarium requires further investigations in light of the breakthrough which has been achieved with mass vaccinations.

Background Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. Methods Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. Results Significantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells. Conclusions Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

Objective: To collect data on vitamin D (25(OH)D) serum levels in a large number of rheumatoid arthritis (RA) patients from different European countries, to investigate their relation with disease activity, disability, quality of life, and possibly to construct a new Patient Reported Outcome (PRO) questionnaire in order to self- estimate if they are at risk for vitamin D insufficiency/deficiency-related clinical implications (D-PRO). Methods: This was a European League Against Rheumatism (EULAR) supported cross-sectional study (project No CLI064) which involved 625 RA patients (mean age 55±11years, mean disease duration 11±9years), 276 age and sex matched healthy subjects, and rheumatologists working in academic institutions or hospital centres, as well as PARE organizations (patient representatives) from 13 European countries. Serum samples for 25(OH)D level measurement were collected during winter time and analyzed in a central laboratory using chemiluminescence immunoassay (DiaSorin). Patient past medical history was recorded. RA patients were provided with three questionnaires: the Rheumatoid Arthritis Impact Diseases score (RAID), the Health Assessment Questionnaire (HAQ), and the new D-PRO questionnaire at the time of 25(OH)D serum sampling. D-PRO questionnaire consisted of three domains, Symptom Risk Score (SRS), Habitus Risk Score (HRS) and Global Risk Score (SRS+HRS=GRS), constructed with items possibly related to vitamin D deficiency. D-PRO was correlated with both clinical and PRO scores. DAS28-CRP was also evaluated. Statistical analysis was performed by non parametric tests. Results: Mean serum concentration of 25(OH)D in RA patients (17.62±9.76ng/ml) was found significantly lower if compared to the levels obtained in matched controls (18.95±9.45ng/ml) (p=0.01), with statistically significant differences among several European countries. Negative correlations were found between 25(OH)D serum levels and DAS28- CRP (p

We read the interesting article by Aungier et al suggesting that targeting proinflammatory signals from the C-terminal fibrinogen-like globe domain (FBG) of tenascin-C (TNC) might provide a viable strategy to treat rheumatoid arthritis (RA).1 The present story and possible developments are really interesting and might link to other recent evidences concerning the same argument. We described for the first time in 1992 the distribution of TNC in normal and pathological synovial tissues from patients with RA and osteoarthritis (OA) by indirect immunofluorescence using specific monoclonal antibodies.2 Tenascin was found in normal synovium just beneath the whole lining cell layer; however, a higher density and spreading pattern of distribution was observed in RA and OA sections, but the possible meaning was unclear at that time. Soon after, these early data were confirmed by others, and several investigations added …

Objectives Fibroblast-to-myofibroblast transition and extracellular matrix overproduction represent progressive events in chronic inflammatory and fibrotic diseases, in which TGFβ1 is one of the key mediators. Phosphodiesterase 4 (PDE4) acts as a proinflammatory enzyme through the degradation of cyclic adenosine monophosphate and it is overexpressed in skin fibroblasts. The study investigated how apremilast (a PDE4 inhibitor) interferes with the intracellular signalling pathways responsible for the TGFβ1-induced fibroblast-to-myofibroblast transition and profibrotic extracellular matrix protein synthesis. Methods Cultured human skin fibroblasts were stimulated with TGFβ1 (10 ng/ml) alone or combined with apremilast (1 and 10 μM) for 4, 16 and 24 h. Other aliquots of the same cells were previously stimulated with TGFβ1 and then treated with apremilast (1 and 10 μM) for 4, 16 and 24 h, always under stimulation with TGFβ1. Gene and protein expression of αSMA, type I collagen (COL1) and fibronectin were evaluated, together with the activation of small mothers against decapentaplegic 2 and 3 (Smad2/3) and extracellular signal-regulated kinase (Erk1/2) proteins. Results Apremilast reduced the TGFβ1-induced increase in αSMA, COL1 and fibronectin gene expression at 4 and 16 h, and protein synthesis at 24 h of treatment in cultured fibroblasts, even for cells already differentiated into myofibroblasts by way of a previous stimulation with TGFβ1. Apremilast inhibited the TGFβ1-induced Smad2/3 and Erk1/2 phosphorylation at 15 and 30 min. Conclusion Apremilast seems to inhibit in vitro the fibroblast-to-myofibroblast transition and the profibrotic activity induced by TGFβ1 in cultured human skin fibroblasts by downregulating Smad2/3 and Erk1/2 intracellular signalling pathways.

Background One of the characteristics of systemic sclerosis (SSc) is an increase in dermal thickness (DT) (1-3). Although the standard method to evaluate the extent of skin involvement is the modified Rodnan skin score (mRSS) (3,4), high frequency ultrasounds (US) and the plicometer skin test (Plicometry) (5-8) are now being used in SSc patients. Objectives The aim of this study was to determine any correlations between mRSS, US and Plicometry during the evaluation of skin impairment in SSc patients. Methods A total of 63 SSc patients (mean age 64±13SD years, mean SSc duration 7±6 years) and 63 healthy subjects (HS) (mean age 64±12SD years) were enrolled. The three methods (mRSS, US and Plicometry) were used to evaluate skin impairment in the seventeen areas of the skin usually evaluated by mRSS (face, fingers, dorsum of hands, forearms, arms, chest, abdomen, thighs, legs and feet) and the total score was calculated, as previously reported (1,3,4,8). Intra-rater reliability of the three techniques was assessed by having the same rater performing 2 consecutive measurements at each skin site. Statistical evaluation was performed by non-parametric tests. Results A significant positive correlation was observed between the three methods used to evaluate DT in the SSc patients (mRSS vs US r=0.64, p 0.05). The intraclass correlation coefficients for mRSS was 0.95, 0.97 for US and 0.96 for Plicometry. Data collection for mRSS took almost 10 minutes, 15 minutes for Plicometry and 20 minutes for US. Conclusion This study demonstrates a significant relationship between mRSS, US and Plicometry in the DT evaluation of SSc patients. The SSc patients had statistically significantly higher values than HS when the 3 techniques were used to evaluate the seventeen skin areas. References [1] Cutolo M, et al. Best Pract Res Clin Rheumatol. 2016;30:670-687. [2] Ruaro B, et al. Arthritis Rheumatol 2015; 67 (suppl 10). [3] Clements P, et al. J Rheumatol 1995;22:1281-5. [4] CzirjakL, et al. Ann Rheum Dis. 2007; 66: 966-9. [5] Hesselstrand R, et al. Rheumatology 2008;47:84-7. [6] Kaloudi O, et al. Ann Rheum Dis. 2010;69:1140-3. [7] Ruaro B, et al. Microvasc Res. 2018;115:28-33. [8] Parodi MN, et al. Br J Rheumatol. 1997;36:244-50. Disclosure of Interests None declared

Background: Systemic sclerosis (SSc) is characterized by immune system alterations, vascular damage and fibrosis (1). Macrophages seem to have an emerging role in SSc, and the characterization of their polarized phenotype, starting from the dichotomic definition of classically activated (M1) or alternatively activated (M2) macrophages, is a recent research topic of interest (2). Objectives: The study investigated a possible imbalance in the distribution of circulating cells expressing M1 and M2 markers in SSc patients (pts) compared to healthy subjects (HSs), and the presence of circulating cells co-expressing M1 and M2 surface markers. Possible correlations between their percentage and selected SSc clinical aspects were investigated. Methods: In the study 55 SSc pts (50 females/5 males, mean age 64±13 yrs), fulfilling the new EULAR/ACR criteria for SSc diagnosis, and 27 age-matched HSs (25 females/2 males, mean age 57±7 yrs) were enrolled after written informed consent. Nailfold videocapillaroscopy (NVC), evaluation of SSc-related antibodies and pulmonary functional tests were performed. In particular, circulating cells belonging to the leukocyte and monocyte populations (CD45+and CD14+cells) were investigated by flow cytometry (FC) using the surface markers characterizing M1 (CD80, CD86, TLR2, TLR4) and M2 phenotypes (CD204, CD206, CD163). Statistical analysis was performed using Mann-Whitney and Kruskal-Wallis tests, and correlations were explored by bivariate Pearson’s analysis. Results: Increased circulating cells showing an M2 phenotype and characterized as CD204+CD206+CD163+cells was observed in SSc pts compared to HSs (p Conclusion: The study identified a circulating cell population expressing both M1 and M2 surface markers, which is increased together with circulating M2 cells in SSc pts, in particular affected by ILD and high PAP, suggesting their possible involvement in the pathogenesis of those disease complications. Further evaluations are in progress. References: [1] Cutolo M, et al. Nat Rev Rheumatol. 2015;11:569-71. 2. Stifano G, et al. Curr Rheumatol Rep. 2016;18:2. Disclosure of Interests: None declared

Background Fibroblast-to-myofibroblast transition and extracellular matrix (ECM) overproduction represent fundamental events in chronic inflammation, that characterise several diseases, including psoriasis (1,2). Transforming growth factor-β1 (TGFβ1), plays an important role as a profibrotic mediator (3). Phosphodiesterases (PDE)4 act as proinflammatory enzymes via degradation of cAMP. PDE4 are expressed in normal skin fibroblasts (Fbs) and overexpressed in psoriatic skin Fbs and myofibroblasts (2). Objectives This study investigated how apremilast (an oral PDE4 inhibitor small molecule used for the treatment of psoriasis and psoriatic arthritis) might interfere with intracellular signalling for the fibroblast-to-myofibroblast transition and the synthesis of profibrotic ECM proteins induced by TGFβ1 in primary cultures of healthy human skin Fbs. Methods Human skin Fbs were isolated from 7 voluntary healthy subjects after signing informed consent and EC approval. The cultured Fbs were stimulated with TGFβ1 10ng/ml alone or in combination with apremilast 1μM and 10μM for 4, 16 and 24 hours. Other aliquots of Fbs were also previously stimulated with TGFβ1 for 4 hours and then treated with apremilast 1μM and 10μM for 4, 16 and 24 hours always in the presence of TGFβ1. Genes and related protein expression of α-smooth muscle actin (αSMA), type I collagen (COL-1) and fibronectin (FN) were investigated by qRT-PCR and Western blotting (WB). Smad proteins (the main signal transducers for receptors of TGFβ1) and extracellular signal–regulated kinases (ERKs), which are implicated in mediating TGFβ1 effects, were investigated by WB after 15, 30 and 60 minutes of apremilast treatment combined with TGFβ1 stimulation. Results Apremilast significantly downregulated the phosphorylation of Smad 2 and 3, at 15 minutes, and that of Erk1/2, after 30 minutes (p Conclusion Apremilast inhibited the fibroblast-to-myofibroblast transition in vitro, as well as the profibrotic activity induced by TGFβ1 in cultured skin Fbs by downregulating specific intracellular signalling pathways Smad 2/3 and Erk 1/2. These results might partially explain some of the downregulating effects on skin Fbs overactivity during treatment of skin lesions of psoriasis and that of psoriatic patients with apremilast. Reference [1] Clarke DL, et al. Fibrogenesis & Tissue Repair 2013, 6:20. 2. Schafer PH, et al. Cell Signal. 2016;28:753-63. 3. Carthy JM. J Cell Physiol.2018;233:98-106. Disclosure of Interests None declared

Background: The fibrosis in systemic sclerosis (SSc) progresses from microvascular alterations, immune system activation, and increased extracellular matrix protein synthesis into the skin and internal organs, primarily mediated by myofibroblasts.1 Myofibroblasts are characterized by a higher expression of α-smooth muscle actin (αSMA) and by the overproduction of type I collagen (COL1) and fibronectin (FN).2 Although myofibroblasts primarily derive from fibroblasts differentiation and transition, circulating fibrocytes represent a further important source.3 Nintedanib is a tyrosine kinase inhibitor that interacts with several inflammatory and profibrotic pathways implicated in the pathogenesis of fibrosis, including those of platelet derived growth factor receptors, vascular endothelial growth factor receptors and fibroblast growth factor receptors.4 Objectives: To investigate, in primary cultures, the effects of nintedanib on the differentiation of circulating fibrocytes and on the profibrotic activity of skin fibroblasts isolated from the same SSc patients. Methods: Circulating fibrocytes and fibroblasts were obtained from peripheral blood and skin biopsies, respectively, from three untreated SSc patients with diffuse skin involvement (mean age 55±6 yrs). To investigate the complete differentiation, fibrocytes were maintained in DMEM at 20% of foetal bovine serum, either with or without treatment with nintedanib at the concentrations of 0.1μM and 1μM for 8 days.5 Fibrocytes were characterized as CD45+CXCR4+COL1+cells, and their percentage was detected by Flow Cytometry analysis.6 Fibroblasts were grown until the 3dr culture passage and then maintained in normal growth medium with or without nintedanib treatment for 4, 24 and 48 hours. The gene expressions of αSMA, COL1 and FN were evaluated by qRT-PCR. Results: Nintedanib reduced the percentage of differentiated SSc fibrocytes (CD45+CXCR4+COL1+cells), already at the concentration of 0.1μM compared with untreated fibrocytes. In cultured SSc skin fibroblasts, nintedanib 1μM downregulated αSMA, COL1 and FN expressions already after 4 hours of treatment, and this effect was also maintained after 24- and 48-hours’ treatment. Nintedanib 0.1μM downregulated the αSMA expression at all timepoints, whereas the downregulation of COL1 and FN expressions was observed at 4 and 24 hours, with no more effect at 48 hours of treatment compared with untreated cells. Conclusion: Initial experiments seem to indicate an evident in vitro concentration-dependent antifibrotic activity of nintedanib on SSc fibrocytes and skin fibroblasts from the same SSc patients; in particular by reducing the fibrocyte differentiation as potential source of myofibroblasts and contrasting the profibrotic activity of already activated fibroblasts/myofibroblasts. These results have translational implications in clinical trials using nintedanib in SSc patients. References: [1] Furue M, et al. Immunol Res 2017;65:790−7. [2] Kendall RT, et al. Front Pharmacol 2014;5:123. [3] Strieter RB, et al. J Leukocyte Biol 2009;88:111−8. [4] Huang J, et al. Ann Rheum Dis 2017;76:1941–8. [5] Cutolo M, et al. Arthritis Res Ther 2018;20:157. Disclosure of Interests: None declared

Background The importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become increasingly recognized, as evidence by incorporation into the 2013 ACR/EULAR clinical classification criteria (1). Clear prognostic and phenotypic associations with cutaneous subtype and internal organ involvement have been extensively described (2). However, little is known about the potential of those autoantibodies to exert a direct pathogenetic role in the disease (3). Objectives The aim of the study is to assess the potential pathogenetic role of anti-topo isomerase I (Topo-I) and anti-centromeric protein B (Cenp B) autoantibodies to induce pro-fibrotic markers in dermal fibroblasts. Methods Dermal fibroblasts were isolated from unaffected and affected skin samples of (n=10) limited cutaneous SSc (LcSSc) patients, from affected skin samples of (n=10) diffuse cutaneous (DcSSc) patients and from (n=20) healthy subjects. Fibroblasts were stimulated with serum-isolated fractions of Topo-I, Cenp B and control IgGs in ratios 1:100 and 1:200 for 24 and 48 hours. Cells were also incubated with 10% SSc Topo-I+, Cenp B+ whole serum and with 10% control serum for 24 and 48 hours. Viability was assessed by MTT test, while apoptosis was assessed by flow cytometry. Activation of pro-fibrotic genes ACTA2, COL1A1 and TAGLN was evaluated by quantitative-real-time-PCR (qPCR), while protein levels alpha-smooth-muscle actin (α-SMA), type-I-collagen (Col-I) and SM22 were assessed by immunocitochemistry (ICC). Results MTT test showed that Cenp B (p Conclusion This study demonstrates the pathogenetic role of antibodies targeting Topo-I and Cenp B to directly induce pro-fibrotic activation of fibroblasts. Therefore, besides the diagnostic and prognostic use of those autoantibodies in SSc, these data justify the importance of therapeutic use of immunosuppressive drugs in the early stages of the disease. References [1] Denton CP. Advances in pathogenesis and treatment of systemic sclerosis. Clin Med (Lond) 2016;16:55-60. [2] Asano Y. Systemic Sclerosis. J Dermatol 2017; doi: 10.1111/1346-8138.14153. [3] Yayla ME, Ilgen U, Duzgun N. An analysis of the relationship between autoantibodies and clinical findings in patients with systemic sclerosis. Turk J Med Sci 2018;48(1):10-15. Disclosure of Interests None declared

Background Systemic sclerosis (SSc) is characterized by early skin impairment (1,2) and the modified Rodnan skin score (mRSS) is the validated method to assess the severity of this impairment. Whilst skin high frequency ultrasound (US) is a relatively new technique to measure dermal thickness (DT) (2-4). Recent findings have reported the important contribution circulating fibrocytes make in the early stage of dermal repair and fibrosis. Indeed, as fibrocytes may be an important source of activated fibroblasts/myofibroblasts, it is reasonable to presume that they could be responsible for the increase in these cells observed in the tissue of SSc patients (5-7). Objectives The aim of this study was to identify any correlation between the modified Rodnan skin score (mRSS), dermal thickness (DT), measured by skin high frequency ultrasound (US) and the percentage of circulating fibrocytes in patients with limited cutaneous systemic sclerosis (lcSSc). Methods After obtaining approval and written informed consent and the Ethics Committee approval 8 lcSSc patients (7 females, 1 male) and 5 (4 females, 1 male) age-matched healthy volunteers (CNT) were enrolled. The lcSSc patients fulfilled the 2013 aCR/EULAR criteria for SSc (8). DT was evaluated by both mRSS and US (18 and 22 MHz probes), in all SSc patients and CNT, in the standard 17 skin areas evaluated by mRSS. The percentage of circulating fibrocytes was obtained by isolating them from the peripheral blood mononuclear cells (PBMCs) in all lcSSc patients and CNTs. Non-parametric tests were used for the statistical analysis. Results The percentage of circulating fibrocytes was positively correlated with DT-US, evaluated by the 22 MHz and the 18 MHz probes (p=0.04 and p=0.03, respectively) and mRSS (p=0.04) in lcSSc patients. Conversely, there was no correlation between these parameters in the CNT group (p>0.05). Conclusion The study demonstrates a significant relationship between DT, evaluated by both mRSS and US and the percentage of circulating fibrocytes in lcSSc patients. This observation may well support the hypothesis that circulating fibrocytes make a crucial contribution to skin fibrosis progression. References [1] Cutolo M, et al. Best Pract Res Clin Rheumatol. 2016;30:670-87. 2. Krieg T, et al. Rheumatology 2009;48:iii14-8. 3. Clements PJ, et al. J Rheumatol 1995;22:1281-5. 4. Ruaro B, et al. Arthritis Rheumatol 2015; 67 (suppl 10). 5. Cutolo M, et al. Arthritis Res ther. 2018;20:157. 6. Brunasso aM, et al. F1000Res. 2016 apr 22;5. doi: 10.12688/f1000research.7986.1. 7. Reilkoff R, et al. Nat Rev Immunol. 2011;11:427-35. 8. van den Hoogen F, et al. Ann Rheum Dis. 2013;72:1745-55. Disclosure of interests None declared

Introduction: Systemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by chronic and progressive tissue and organ fibrosis with broad patient-to-patient variability. Some risk factors are known and include combination of persistent Raynaud's phenomenon, steroid hormone imbalance, selected chemicals, thermal, or other injuries. Endogenous and/or exogenous environmental trigger/risk factors promote epigenetic mechanisms in genetically primed subjects. Disease pathogenesis presents early microvascular changes with endothelial cell dysfunction, followed by the activation of mechanisms promoting their transition into myofibroblasts. A complex autoimmune response, involving innate and adaptive immunity with specific/functional autoantibody production, characterizes the disease. Progressive fibrosis and ischemia involve skin and visceral organs resulting in their irreversible damage/failure. Progenitor circulating cells (monocytes, fibrocytes), together with growth factors and cytokines participate in disease diffusion and evolution. Epigenetic, vascular and immunologic mechanisms implicated in systemic fibrosis, represent major targets for incoming disease modifying therapeutic approaches. Areas covered: This review discusses current understanding and new insights of SSc pathogenesis, through an overview of the most relevant advancements to present aspects and mechanisms involved in disease pathogenesis. Expert opinion: Considering SSc intricacy/heterogeneity, early combination therapy with vasodilators, immunosuppressive and antifibrotic drugs should successfully downregulate the disease progression, especially if started from the beginning.

Background:Among the cells involved in the inflammatory process of rheumatoid arthritis (RA) [1], macrophages play a key role through their capacity to polarize into “classically” or “alternatively” activated phenotypes (M1 or M2) and making macrophages important players for the inflammatory cascade or for the anti-inflammatory reaction, respectively [2]. CTLA4-Ig fusion protein (abatacept) has been shown to contribute to macrophage shift from M1 to M2 [3].Objectives:We aimed to investigate the effects of abatacept to induce the polarization from the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype in cultured human macrophages obtained from RA patients’ and healthy subjects’(HS) circulating monocytes.Methods:Cultured monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of three early RA patients and ten HS, after signing informed consent and Ethics Committee approval. Cells were treated with phorbol myristate acetate (PMA) [5ng/ml] for 24 hours (hrs) to induce their differentiation into monocyte-derived macrophages (MDMs). Therefore, cultured HS MDMs were stimulated with lipopolysaccharides [LPS, 1mg/mL] for 4hrs [4] in order to induce their polarization into a pro-inflammatory M1 phenotype and then treated or not with abatacept at the concentrations of 100mg/mL and 500mg/mL for 3, 12, 24 and 48hrs. Cultured RA MDMs, were directly treated with abatacept as previous described. Cultured HS and RA MDMs without any pro-inflammatory stimuli and abatacept treatment were used as respective control.The transition of MDMs from M1 to M2 phenotype was evaluated through gene expression and protein synthesis of M2 macrophage markers, namely scavenger receptors (CD163 and CD204), and mannose receptor-1 (CD206) by quantitative real-time polymerase chain reaction (PCR) and by Western blotting. The statistical analysis evaluation was carried out by GraphPad Prism 8 analysis software using the Wilcoxon non-parametric t-test. Any p-value lower than 0.05 was considered as statistically significant. Results were indicated as median±standard deviation (SD).Results:In cultured RA MDMs (three cases), abatacept upregulated the gene expression of all investigated M2 markers, specifically after 12hrs of treatment with the concentration of 100mg/mL. In these cells, abatacept upregulated only the CD204 protein synthesis with more evidence at 24hrs of treatment and with the 500mg/mL concentration. In cultured HS MDMs (ten cases), abatacept upregulated the gene expression of M2 markers, significantly for that of CD206 [at 3hrs with 100mg/mL concentration, p= 0.0312] and CD163 [at 12hrs with 500mg/mL concentration, p= 0.0312]. Moreover, in these cells, abatacept significantly upregulated the protein synthesis of CD206 [at 48hrs with 500mg/mL concentration, p= 0.0195] and CD204 [at 24hrs with 100mg/mL concentration, p= 0.0156; both at 24 and 48hrs with 500mg/mL concentration, p= 0.0234].Conclusion:Preliminary data seem to indicate that abatacept can promote the in vitro shift from the M1 into the M2 macrophage phenotype, by upregulating specific markers (CD163, CD204, CD206) in cultured M1-MDMs from RA patients and in M1 macrophages induced from HS.References:[1]McInnes IB, et al. N Engl J Med 2011;365:2205–19.[2]Fujii M, et al. Biochem Biophys Res Commun. 2013;438(1):103-9.[3]Cutolo M, et al. Arthritis Res Ther. 2009;11:R176.[4]Pelegrin P., Surprenant, A. EMBO J. 2009 Jul 22; 28(14): 2114–2127.Disclosure of Interests:Samuele Tardito: None declared, Stefano Soldano: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene.

Background:Fibroblast-to-myofibroblast transition is one of the fundamental steps involved in the fibrotic process that characterise systemic sclerosis (SSc) [1]. Myofibroblasts are α-smooth muscle actin (αSMA) positive cells that contribute to fibrosis through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, primarily fibronectin (FN) and type I collagen (COL1) [2].Among the cells involved in the fibrotic process of SSc, circulating fibrocytes seem to have an emerging role as an important source of fibroblasts and myofibroblasts [3].Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis (4). Nintedanib was recently demonstrated to have a beneficial effect in patients with interstitial lung disease (ILD) associated with SSc (5).Objectives:To investigate nintedanib effect in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.Methods:Circulating fibrocytes were obtained from 14 SSc patients (mean age 64±14 years), who fulfilled the 2013 ACR/EULAR criteria for SSc and that underwent complete disease staging in a day-hospital setting at the Rheumatology Division of Genoa University. Five age-matched healthy subjects (HSs) were also analysed. All SSc patients and HSs signed the informed consent and the local EC approved the study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and plated on FN-coated dishes. After overnight culture, non-adherent cells were removed, and adherent cells were maintained in growth medium for 8 days (T8) to obtain fibrocytes [6]. T8-cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1μM and 1μM for 3 and 24 hours. Fibroblast specific protein-1 (S100A4) and αSMA, as markers of fibroblast/myofibroblast phenotype, together with COL1 and FN, were investigated by qRT-PCR and Western blotting. Non-parametric Mann-Whitney and Wilcoxon tests were used for the statistical analysis.Results:Significantly elevated gene and protein expressions of αSMA, S100A4, COL1 and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p+ILD+) or Scl70 negative patients without ILD (Scl70-ILD-). Significant αSMA, S100A4, COL1 and FN gene expressions were found in fibrocytes from Scl70+ILD+ compared to HS fibrocytes (αSMA p+ILD+patients showed a more significant gene expression of fibroblasts/myofibroblasts markers compared to Scl70-ILD-patients (pNintedanib reduced the gene and protein expression of αSMA, COL1 and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated αSMA, S100A4, COL1 and FN gene expression (all p+ILD+fibrocytes, whereas only that of S100A4 and FN was significantly downregulated (p-ILD- fibrocytes compared to untreated cells.Conclusion:Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+SSc patients.References:[1]Cutolo M et al. Exp Rev Clin Immunol. 2019;15:753-64.[2]Van Caam A et al. Front. Immunol. 2018;9:2452.doi:10.3389/fimmu.2018.02452.[3]Distler JH et al. Arthritis Rheumatol. 2017;69:257-67.[4]Distler O et al. New Eng J Med. 2019; 380:2518-28.[5]Maher TB et al. Arthritis Rheumatol.2020.doi:10.1002/art.41576.[6]Cutolo M et al. Arthritis Res Ther. 2018;20:157.doi:10.1186/s13075-018-1652-6.Acknowledgements:We thank Stefano-Lutz Willing for the scientific support through the study.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Emanuele Gotelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli: None declared, Carlotta Schenone: None declared, Greta Pacini: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene

Background:muscle actin (aSMA)+cells involved in the overproduction of extracellular matrix proteins, primarily fibronectin (FN) and type I collagen (COL1) at the level of damaged tissues (1). These cells may originate from different cell types including fibroblasts, endothelial and epithelial cells, and fibrocytes (1). Circulating fibrocytes are bone marrow progenitor cells expressing specific markers of hematopoietic (CD34, CD45, and MHC class II) and stromal cells (COL1 and COL3), chemokine receptors (CCR2, CCR7), and CXCR4 (2). CXCR4 regulates fibrocyte migration into injured tissues allowing their differentiation into fibroblasts/myofibroblasts (2).In vitro, fibrocytes differentiate from circulating CD14+monocytes showing an antigen-presenting capability through the expression of HLA-DR and costimulatory molecule CD86 (2). CTLA4-Ig fusion protein (abatacept) interacts with CD86 on cell surface of antigen presenting cells (APCs), such as macrophages and endothelial cells (3,4).Objectives:To investigate the possible effect of CTLA4-Ig treatment on cultured human fibrocytes and skin fibroblasts isolated from the same systemic sclerosis patients (SSc pts).Methods:Fibrocytes isolated from the peripheral blood mononuclear cells of SSc pts and healthy subjects (HSs) were cultured on fibronectin-coated plates in DMEM at 20% of FBS; for further 8 days (T8) to allow their complete differentiation. Differentiated fibrocytes were maintained in growth medium or treated with CTLA4-Ig at different concentrations (10, 50, 100, and 500μg/ml) for 3 hours. Fibroblasts were isolated from the skin biopsies of the same patients and HSs, cultured until the 3rdpassage in RPMI at 10% FBS and then treated with CTLA4-Ig for 24 and 48 hours. Fibrocytes were characterized as CD45+CXCR4+COL1+cells and the expression of CD86 and HLA-DR was also evaluated. The gene expression of aSMA, COL1, CXCR4, TGFb1 and CD86 was investigated by quantitative real-time polymerase chain reaction in cultured fibrocytes and skin fibroblasts. In cultured skin fibroblasts, COL1 and fibronectin synthesis was evaluated by Western blotting.Results:Treatment with CTLA4-Ig for 3 hours significantly downregulated aSMA and COL1 gene expression in cultured SSc fibrocytes at T8 (pConclusion:Due to their high expression of CD86, circulating fibrocytes seem to be more responsive to CTLA4-Ig treatment than the skin fibroblasts isolated from the same SSc patient.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64.[2]Bucala R. Mol Med.2015;2:S3-5.[3]Cutolo M et al. Clin Exp Rheumatol. 2015;33:250-4.[4]Brizzolara R et al. J Rheumatol. 2013;40:738-40.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Elisa Alessandri: None declared, Carmen Pizzorni: None declared, Greta Pacini: None declared, Federica Goegan: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

Background:Alternatively activated (M2) macrophages seem to play a role in the fibrotic process of systemic sclerosis (SSc) as potential inducers of tissue fibrosis through their secretion of specific cytokines and chemokines, such as interleukin-10 (IL-10), macrophage derived chemokine (CCL-22) and pro-fibrotic metalloproteases (i.e. MMP9) (1-3).Objectives:To investigate the presence of circulating cells belonging to the monocyte lineage showing an M2 phenotype in SSc patients (pts) and possible correlation with the clinical parameters of the disease. Moreover, to investigate if the treatment of cultured monocytes isolated from healthy subjects with serum derived from early SSc pts may induce theirin vitropolarization into M2 macrophages.Methods:Fifty female SSc pts (mean age 64±13 yrs), fulfilling the EULAR/ACR criteria, and 27 gender-matched healthy subjects (HSs, mean age 57±7 yrs) were considered at the Rheumatology Division of Genoa University after written informed consent. Nailfold videocapillaroscopy (NVC), serum SSc-related antibodies and skin involvement were investigated. Circulating cells belonging to the monocyte populations (CD45+and CD14+cells) were characterised by flow cytometry using specific surface markers of M2 phenotypes (CD204, CD206, CD163). Each SSc pt had been under stable treatment regimen for at least six months. Cultured monocytes, isolated by negative selection from peripheral blood mononuclear cells (PBMCs) of 8 HSs, stimulated for 48 hrs with 10% of serum of lcSSc pts with “Early” NVC pattern, as well as serum of dcSSc pts with “Active” and “Late” NVC patterns. Cultured monocyte human cell line (THP1) was differentiated into macrophages (5ng/ml of phorbol myristate acetate) and then stimulated with SSc sera. The expression of CD204, CD206 (M2 markers) and CD68 was investigated by immunocytochemistry, whereas MMP9 secretion was investigated by zymography. Statistical analysis was performed using Mann-Whitney and Kruskal-Wallis tests, and correlations were explored by bivariate Pearson’s analysis.Results:In SSc pts the percentage of circulating M2 cells (CD14+CD204+CD163+CD206+cells) was significantly increased compared to both HSs and SSc pts not under immunosuppressive treatment (pConclusion:The study confirmed that SSc pts are characterized by a significant increase of circulating M2 cells, suggesting their possible involvement in the pathogenesis of the disease. Interestingly, results insinuate that sera from SSc patients already in an “Early” NVC condition (sera known to contains specific profibrotic molecules such as cytokines, growth factors like TGFb1 or endothelin-1) seem able to inducein vitroa profibrotic M2 macrophage phenotype.References:[1]Cutolo M et al. ExpRevClin Immunol. 2019;15:753-64.[2]Stifano G et al. Curr Rheumatol Rep. 2016; 18:2. doi: 10.1007/s11926-015-0554-8.[3]Medeiros NI et al. Parasite Immunol. 2017;39: doi: 10.1111/pim.12446.Disclosure of Interests:Stefano Soldano: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Massimo Patanè: None declared, Emanuele Gotelli: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Greta Pacini: None declared, Federica Goegan: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

Background:Exosomes generated great resonance in the last few years due to their important roles in different biological pathways and diseases, including systemic sclerosis (SSc) (1). They are lipid-like nanovesicles containing biomarkers, such as proteins, lipids, macromolecules and nucleic acids, including microRNA (miRNA) (2). Exosomes are implicated in intercellular communication by fusing and releasing their cargo into the target cells (3).Objectives:In the present study, we evaluated the potential of exosomes deriving from plasma of SSc patients or generating from cultured SSc fibroblasts to drive the fibrotic signaling in the disease.Methods:Exosomes were isolated from plasma of n=10 SSc patients and from n=10 control subjects. Exosomes were also purified from cell culture supernatants of SSc fibroblasts and of control fibroblasts. Exosome size and concentration were assessed by Nanosight Particle Tracking Analysis (NTA) and by transmission electron microscopy (TEM). The content of anti-fibrotic (let-7a, 146a, 200a, 223a) and pro-fibrotic (150, 155) miRNAs was assessed in all the plasma-derived and cell culture-derived exosome populations by semiquantitative real time PCR. Finally, isolated exosomes were used to stimulate control dermal fibroblasts in culture. Gene expressions (COL1A1, ACTA2 and TAGLN) were assessed by quantitative real time PCR (qRT-PCR) and protein levels (type-I-collagen, α-SMA and SM22) by immunofluorescence (IF).Results:Exosomes isolated from SSc plasma samples showed higher concentration (3.3x1010±1.1x1010particles/mL) compared to those isolated from control plasma ones (1.5x1010±0.4x1010particles/mL) (p10±0.2x1010particles/mL) than in control ones (0.4x1010±0.1x1010particles/mL) (pConclusion:This study demonstrates that plasma from SSc patients contains higher concentration of exosomes compared to plasma from control subjects and SSc-derived exosomes contain specific pro-fibrotic miRNA signatures that can induce myofibroblast differentiationin vitro. These results suggest that exosomes could be fibrotic drivers towards non-affected areasin vivo, and they might represent novel targets for precision medicine treatments in SSc.References:[1]Zhu T, Wang Y, Jin H, Li L. The role of exosome in autoimmune connective tissue disease. Ann Med. 2019 Mar;51(2):101-108.[2]Wermuth PJ, Piera-Velazquez S, Rosenbloom J, et al. Existing and novel biomarkers for precision medicine in systemic sclerosis. Nat Rev Rheumatol. 2018 Jul;14(7):421-432.[3]Colletti M, Galardi A, De Santis M, et al. Exosomes in Systemic Sclerosis: Messengers Between Immune, Vascular and Fibrotic Components? Int J Mol Sci. 2019 Sep 4;20(18). pii: E4337.Disclosure of Interests:Claudio Corallo: None declared, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha, Stefano Soldano: None declared, Enrico Selvi: None declared, Francesca Bellisai: None declared, Nicola Giordano: None declared

Background:Systemic sclerosis (SSc) is a chronic connective disease characterized by microvascular alterations, dysregulated immune response and fibrosis [1,2]. Myofibroblasts are alpha-smooth muscle actin (alphaSMA)+cells and play a crucial role in fibrosis, through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, in particular fibronectin (FN) and type I collagen (COL1) [3]. Despite myofibroblasts primarily derive from resident fibroblasts transition and differentiation, another important source is represented by circulating fibrocytes [4]. Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis [5].Objectives:To investigate the possible effects of nintedanib in contrasting the ability of cultured mature fibrocytes from SSc patients to differentiate into profibrotic myofibroblasts.Methods:Circulating fibrocytes were obtained from peripheral blood mononuclear cells isolated from 5 limited cutaneous SSc patients (mean age 68 +/- 10 years) and then plated on FN-coated tissue culture dishes in growth medium (DMEM at 20% of fetal bovine serum, 1% of penicillin-streptomycin and 1% L-glutamine), to allow the adhesion of fibrocyte precursors. Adherent cells were maintained in growth medium for 8 days in order to allow their differentiation into fibrocytes. Differentiated fibrocytes were treated with nintedanib at the concentrations of 100nM and 1000nM for 3 and 24 hours (hrs) or maintained in growth medium without any treatment. The differentiation of fibrocytes into myofibroblasts was determined evaluating the gene expression of alphaSMA, fibroblast specific protein-1 (S100A4) COL1, FN and CXCR4 by quantitative real-time polymerase chain reaction, and the protein synthesis of alphaSMA, COL1 and FN by western blotting.Results:Nintedanib inhibited alphaSMA and S100A4 gene expression already at the concentration of 100nM in cultured fibrocytes and after 3 hrs of treatment, when compared with untreated cells. Furthermore, both concentrations of nintedanib (100nM and 1000nM) reduced the gene expression of COL1 and FN, whereas only 100nM downregulated the CXCR4 gene expression. At protein level, nintedanib 100nM and 1000nM reduced the synthesis of alphaSMA and COL1 after 24 hrs of treatment, whereas FN synthesis was reduced only by the nintedanib concentration of 1000nM.Conclusion:The preliminary results show that nintedanib may inhibit thein vitrotransition of SSc fibrocytes into myofibroblasts and their profibrotic activity, through the reduction of specific myofibroblast phenotype markers and ECM protein production. The results seem to suggest fibrocytes as further possible target of the antifibrotic action of nintedanib in SSc.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64 2. Barsotti S et al. Clin Exp Rheumatol. 2016;34(Suppl.100):S3-S13 3. Wynn TA et al. Nat Med. 2012;18:1028-40. 4.Distler JHW et al. Arthritis Rheumatol. 2017;69:257-67 5.Hilberg F et al. Cancer Res. 2008;68:4774-82.Disclosure of Interests:Stefano Soldano: None declared, Giulia Martinelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Massimo Patanè: None declared, Emanuele Gotelli: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

Background and aim Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease; the clinical manifestations are correlated with continuum multiarticular synovitis, cartilage and bone damage, and defeat of joint function, that causes disability. Involvement of internal organs is also frequent. Between the inflammatory cells involved in RA, macrophages play a key role. These cells can polarize in different phenotype and mediate the immune/inflammatory reaction as well as the reparatory phase when possible. The properties of these cells are mediate by the body's environmental factors. In this systematic review, all English-speaking articles concerning the role of M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages in RA were systematically reviewed and categorized according to their polarized-function in RA, especially in the synovial tissue. Analyses of the endogenous molecules and the drugs that could modulate M1 and M2 activity in RA were achieved. Methods A sensitive search was developed in Pubmed, Web of Science, Ovid Med-Line, Embase Database and Science Direct Database (la both from Elsevier) to identify articles to increase the highlighting on the role of macrophages M1 and M2 in RA using the following terms: ((M1 AND M2) AND Rheumatoid Arthritis). All selected papers were read and discussed by two independent reviewers. The selection process was based on title, abstract and full text level. Relevant data were extracted and analyzed using a standardized template designed for this review. Results In total 39 resulting articles were selected and categorized according to description of M1/M2's role in RA. Data from humans, mice and rats were subcategorized, thus in every section were highlighted the contribute, in peripheral blood and synovial tissue, of both polarized macrophages; section for endogenous molecules and drugs that favor the switch from M1 to M2 macrophages were carried out. The most evinced relevant results, were that in RA blood and in the synovial tissue, there isn't a clear distinction phase with M1 or M2 macrophages (by membrane marker analysis); rather there is M1 and M2 subset disequilibrium and by deeply analyses of mRNA gene and cytokine produced, it emerged that a non-coherent expression inner marker match with membrane molecules, and also the tissue section can define the marker expressed. Conclusion This systematic review emphasizes that the rigid classical subdivision of M1 and M2 macrophages, as well as the different samples' results comparison, might be questionable. In addition, it is suggested, when taking samples from RA patients, to carefully consider their therapies in order to analyze the M1 and M2 macrophages behavior without drug influence. In line with the advances in M1 and M2 knowledge, and the progression in the single-cell methodologies by identification of individual cell molecular markers, therapeutic approaches seem possible to favor the anti-inflammatory macrophage response in RA (e.g. M2 polarization).

The objective is to detect any possible correlation between the modified Rodnan skin score (mRSS) and dermal thickness (DT) measured by skin high-frequency ultrasound (US) and the percentage of circulating fibrocytes in patients with limited cutaneous systemic sclerosis (lcSSc). Eight lcSSc patients and five healthy subjects (control group, CNT) were enrolled. The skin involvement was evaluated by mRSS and US (18 and 22 MHz probes) in all 13 subjects in the 17 standard skin areas evaluated by mRss. Circulating fibrocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of all lcSSc patients and the CNT group to analyze their percentage at baseline time (T0) when the experiments started with PBMCs’ isolation and collection and after 8 days of culture (T8). Non-parametric tests were used for the statistical analysis. A positive correlation between the percentage of circulating fibrocytes at T0, mRSS (p = 0.04 r = 0.96), and DT-US, evaluated by the 22 MHz and the 18 MHz probes (p = 0.03, r = 0.66 and p = 0.05, r = 0.52, respectively), was observed in lcSSc patients. Conversely, at T8, there was no correlation (p > 0.05) between these parameters in lcSSc group. In the CNT group, no correlations between mRSS or DT-US and the percentage of circulating fibrocytes were observed both at T0 and T8. The study shows the presence of a significant relationship between the percentage of circulating fibrocytes and DT, as evidenced by both mRSS and US, in limited cutaneus SSc. This observation may well suggest the reasonable hypothesis of a crucial contribution of circulating fibrocytes to skin fibrosis progression, which might be considered as further biomarkers.

Background In the pathogenesis of systemic sclerosis (SSc), the immune cell activation is an important event that includes alteration in the macrophage polarisation.1 Macrophages may polarise into classically activated (M1), which are characterised by the expression of specific markers such as Toll-like receptors (TLR2 and 4) and costimulatory molecules (CD80 and CD86), or alternatively activated (M2), which are characterised by the expression of specific phenotype markers such as mannose receptor-1 (CD206) and macrophage scavenger receptors (CD204 and CD163). M2 are present in the circulation and in the skin infiltrate of SSc patients (pts), where they seem to contribute to fibrosis.2–4 Objectives To characterise circulating M2 monocytes/macrophages from SSc pts and healthy subjects (HSs) by their co-expression of CD204, CD163 and CD206, as well as cells expressing both M1 and M2 phenotype markers. Methods Fifty-eight SSc pts (54 females/4 males, mean age 63±13 years), fulfilling the new EULAR/ACR criteria for SSc, and 27 age-matched HSs were consecutively enrolled after Informed Consent was obtained. Peripheral blood was collected and the antibodies CD14-APC-Vio770 and CD45-VioGreen were used to identify the monocyte/macrophage lineage; CD204-VioBright-FITC, CD163-PE-Vio770 and CD206-PeerCP-Vio700 were used to characterise the M2 phenotype, whereas CD80-APC, CD86-VioBlue, TLR4-PE and TLR2-PE-Vio615 were used to characterise the M1 phenotype (Miltenij Biothech). Flow Cytometry analysis was performed using Navios Flow Cytometer and the related Navios analysis software (Beckman Coulter). Results In the CD14+cell subset (monocytes), the CD14+CD163+CD206+CD204+cell percentage was significantly increased in SSc pts compared to HSs (p=0.02). Inside the CD14+CD163+CD206+CD204+monocytes/macrophages a subset of cells co-expressing also TLR4, CD80 and CD86 was detected. This mixed population (M2/M1) of cells was significantly increased in SSc pts compared to HSs (p=0.003). At the same time, circulating monocytes/macrophages showing a full M2 phenotype and characterised as CD204+CD163+CD206+cells were investigated independently of the expression of CD14, and they also resulted significantly increased in SSc pts compared to HSs (p In the CD204+CD163+CD206+cell subset (M2), the percentage of cells expressing also TLR4, CD80 and CD86 (M1) was significantly increased in SSc pts compared to HSs (p Conclusions These results describe for the first time a subset of circulating cells belonging to the monocyte/macrophage lineage with a mixed phenotype, which are characterised by the expression of both M1 and M2 surface markers. These cells were observed to be increased in the peripheral blood of SSc pts compared to HSs, suggesting their possible role in the pathogenesis of the disease. References [1] Stifano G, et al. Curr Rheumatol Rep2016;18:2. [2] Higashi-Kuwata N, et al. Arthritis Res Ther2010;12:R128. [3] Wynn TA, et al. Immunol Rev. 2016;44:450–62. [4] Christmann RB, et al. Arhtitis Rheum. 2011;63:1718–28. Disclosure of Interest None declared

Background In systemic sclerosis (SSc), the transition of fibroblasts into profibrotic myofibroblasts contributes to fibrosis through their over-production of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated by the activation of fibrogenic intracellular signalling transduction molecules, including Erk1/2 and Akt kinases.1,2 Selexipag and its active metabolite (ACT-333679) are prostacyclin receptor agonists which leads to vasodilation in the pulmonary circulation and mainly used in the treatment of pulmonary arterial hypertension. Objectives To investigate the possible effects of selexipag and ACT-333679 in downregulating the profibrotic activity of cultured SSc fibroblasts/myofibroblasts and the fibrogenic signalling molecules involved in the process. Methods After obtaining Ethics Committee (EC) approval and informed consent, fibroblasts isolated from skin biopsies of 6 SSc patients (mean age 63±13 years) were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 µM to 0.3 µM) or ACT-333679 (from 10 µM to 0.1 µM) for 48 hours. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by Western blotting and qRT-PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679 treated cells at 15 and 30 min as well as at 48 hours by Western botting. Results Selexipag and ACT-333679 significantly reduced the protein synthesis and the gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, primarily at the concentrations of 3 µM and 0.3 µM for selexipag and 1 µM and 0.1 µM for ACT-333679 (p Of note, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt kinases already after 30 min of treatment in cultured SSc fibroblasts/myofibroblasts, and this effect was further maintained at 48 hours from treatment (p Conclusions For the first time selexipag and mainly its active metabolite ACT-333679 were found to interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts, possibly through the downregulation of fibrogenic Erk1/2 and Akt intracellular signalling molecules. References [1] Ho YY, et al. Nature2014;10:390–402. [2] Distler JHW, et al. Arthrit Rheumatol2017;69:257–67. [3] Gatfield J, et al. J Pharmacol Exp Ther. 2017;362:186–99. Disclosure of Interest M. Cutolo Grant/research support from: Actelion, Bristol-Mayer Squibb, Celgene, B. Ruaro: None declared, P. Montagna: None declared, R. Brizzolara: None declared, A. Trombetta: None declared, S. Scabini: None declared, P. P. Tavilla: None declared, A. Parodi: None declared, C. Corallo: None declared, N. Giordano: None declared, S. Paolino: None declared, C. Pizzorni: None declared, A. Sulli: None declared, V. Smith: None declared, S. Soldano: None declared

Background Skin fibroblasts (SFs) are involved in the excessive production of extracellular matrix (ECM) proteins which characterises fibrosis in systemic sclerosis (SSc).1 Myofibroblasts which are characterised by a higher expression of pro-fibrotic molecules (a-SMA: alpha-smooth muscle actin; S100A4: fibroblast-specific protein-1) as well as by the over-production of ECM proteins (FN: fibronectin; collagens type I and III), may originate from the activation and differentiation of resident fibroblasts after multiple profibrotic stimuli.2 CTLA4-Ig interacts with the cell surface costimulatory molecule CD86 and can downregulate the target cell activation.3 Objectives To evaluate CD86 expression and the in vitro effects of CTLA4-Ig on skin fibroblasts (SFs). Methods Skin biopsies were obtained from 8 “limited” cutaneous SSc patients (treated only with vasodilators, mainly cyclic prostanoids) and 4 healthy subjects (HSs), after EC and patient informed consent. After 8 days (T8) of culture, SFs obtained from biopsies were treated for 24 and 48 hours, in the absence or in the presence of CTLA4-Ig (10, 50, 100 and 500 micrograms/ml). Evaluation of CD86 expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, also human macrophages obtained from PBMCs of SSc patients, were cultured. The statistical analysis was carried out by the non-parametric Mann-Whitney U test. Results Cultured SSc fibroblasts showed a very low gene expression level of CD86, compared to cultured macrophages of SSc patients, taken as positive control for CD86 expression (99% less). Therefore, cultured SSc fibroblasts treated for 24 hours and for 48 hours with CTLA4-Ig (10, 50, 100 and 500 micrograms/ml) did not show any significant modulation in the gene expression levels of CD86, compared to untreated fibroblasts (CNT). Interestingly, cultured HSs fibroblasts treated with CTLA4-Ig for 24 hours and for 48 hours showed a significant decrease in the gene expression of CD86, limited to the highest dose (500 micrograms/ml), compared to CNT (0.16% and 0.64% less, respectively) (p Conclusions In the present short-term study (24 and 48 hours), no significant effects at qRT-PCR resulted after CTLA4-Ig treatment of cultured SSc SFs. The result might arise from a limited expression of CD86, as consequence of a retained advanced differentiation of the the SSc fibroblasts. On the contrary, a significant reduction of CD86 expression on HSs fibroblasts treated with CTLA-4Ig was observed. References [1] Asano Y. J Dermatol2017. Epub ahead of print. [Review]. [2] Hin ZB, Phan SH, et al. Am J Pathol2007;170:1807–16. [3] Cutolo M, et al. Arthritis Res Ther2009;11:176–85. Acknowledgements Disclosure of Interest M. Cutolo Grant/research support from: BMS, Actelion, Celgene, Boehringer, P. Montagna: None declared, S. Soldano: None declared, A. C. Trombetta: None declared, P. Contini: None declared, B. Ruaro: None declared, A. Sulli: None declared, S. Scabini: None declared, E. Stratta: None declared, R. Brizzolara: None declared

Background Systemic Sclerosis (SSc) is an autoimmune disease characterised by progressive fibrosis of the skin and internal organs, coupled to widespread vascular pathology.1 The pathogenesis is still poorly understood and there is no effective treatment for the fibrotic process.1 Relaxin is a potent anti-fibrotic hormone that has been tested in the past to ameliorate skin, lung and kidney fibrosis in SSc, but the results remain controversial.2 Objectives The aim of the study is to evaluate the presence of mutations in RXFP1 gene (encoding the relaxin receptor RXFP1/LGR7), and to assess mRNA and protein levels of the receptor in dermal fibroblasts of SSc patients, in order to understand the clinical inefficacy of relaxin-based anti-fibrotic treatments in the disease. Methods Fibroblasts were isolated from unaffected and affected skin samples of (n=20) of limited cutaneous SSc (LcSSc) and from (n=20) affected skin of diffuse cutaneous SSc (DcSSc) patients. Fibroblasts derived from healthy subjects were used as controls. Sequencing of exonic target regions of interest for gene RXFP1 was performed coupled with mRNA transcript variant analysis. RXFP1/LGR7 mRNA and protein levels were assessed by quantitative-real-time-PCR (qPCR) and by immunocitochemistry (ICC) in cultured SSc and healthy fibroblasts. Finally, synthesis of collagen and alpha-smooth-muscle actin (a-SMA) of transforming-growth-factor-beta-1 (TGF-β1) induced fibroblasts were assessed after 24 hours pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the relaxin receptor RXFP1/LGR7). Results Sequencing of RXFP1 gene showed no relevant (single nucleotide polymorphisms) SNPs or small insertions and deletions (InDels) in affected LcSSc/DcSSc fibroblasts. No relevant mutations were found in unaffected LcSSc and healthy fibroblasts as well. However, alternatively spliced transcript variants encoding multiple isoforms were observed for this gene in all the fibroblast populations. The total RXFP1 mRNA levels resulted upregulated (p Conclusions The absence/altered expression of relaxin receptor RXFP1/LGR7 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease. The exclusion of RXFP1 gene mutations could lead to the hypothesis that the presence of receptor splice variants could exert a dominant negative effect on the wild type isoform in terms of maturation, and subsequent trafficking to the cell surface, resulting in loss of function. References [1] Denton CP. Advances in pathogenesis and treatment of systemic sclerosis. Clin Med (Lond)2016;16:55–60. [2] McVicker BL, Bennet RG. Novel Anti-fibrotic therapies. Front Pharmacol2017;8:318. Disclosure of Interest None declared

Background Circulating fibrocytes (CFs) are progenitor cells derived from bone marrow, expressing markers of both hematopoietic cells (CD45, MHC class II) and stromal cells (collagen I and III), together with the chemokine receptors, which regulate their migration into inflammatory lesions (CXCR4, CCR2, CCR7).1 CFs can migrate into SSc-affected tissues and can differentiate into fibroblasts/myofibroblasts.2 CFs express the CD86 (B7.2) costimulatory molecule and the adhesion molecules CD11a, CD54, ICAM-1, and CD58. The fusion molecule CTLA4-Ig interacts with CD86 and can downregulate the target cell.3 Objectives To study the in vitro effects of CTLA4-Ig on cultured CFs. Methods CFs were obtained from the peripheral blood samples of 8 “limited” cutaneous SSc patients (treated only with vasodilators, mainly cyclic prostanoids) and from 4 healthy subjects (HSs). CFs were characterised by fluorescence-activated cell sorter analysis (FACS), at basal time (T0) and after 8 culture days (T8), for CD45, collagen type I (COL I), CXCR4, CD14, CD86, and HLA-DRII expression. T8-cultured CFs were treated for 3 hours in the absence or in the presence of CTLA4-Ig (10, 50, 100 and 500 micrograms/ml). Quantitative real-time polymerase chain reaction (qRT-PCR) for CD86, COL I, IL1b, TGFb, αSMA, S100A4, CXCR2, CXCR4, CD11a were performed. The statistical analysis was carried out by the nonparametric Mann-Whitney U test. Skin samples for fibroblast (SFs) cultures were obtained from the same patients after EC and patient informed consent. Results At qRT-PCR, T8-SSc fibrocytes, in the absence of CTLA4-Ig treatments, showed higher CD86 expression levels compared to HSs fibrocytes. Similarly also αSMA, S100A4, TGFb and COL I gene expression resulted higher in SSc fibrocytes compared to HSs. After CTLA4-Ig treatments, only in SSc fibrocytes, the αSMA and COL I gene expression resulted significantly decreased (p Conclusions Circulating fibrocytes from patients affected by limited cutaneous SSc seem to be responsive and downregulated after in vitro CTLA4-Ig treatments, suggesting a possible antifibrotic effect on progenitor cells before their final homing and differentiation in active myofibroblasts. Fibroblasts from the same patient do not show the same expression of target molecules and reactivity to CTLA4-Ig. References [1] Bucala R. Mol Med2015;2:S3–5. [2] Keeleya EC, et al. The International Journal of Biochemistry & Cell Biology2010;42:535–42. [3] Cutolo M, et al. Arthritis Res Ther2009;11:176–85. Disclosure of Interest M. Cutolo Grant/research support from: BMS, Actelion, Celgene, Boehringer, P. Montagna: None declared, S. Soldano: None declared, A. C. Trombetta: None declared, B. Ruaro: None declared, P. Contini: None declared, A. Sulli: None declared, S. Scabini: None declared, E. Stratta: None declared, R. Brizzolara: None declared

Objectives To assess the validity of the rheumatoid arthritis impact of disease (RAID) for measuring disease activity of rheumatoid arthritis (RA) and to determine cut-off values for defining the disease activity states. Methods A total of 622 RA patients from an European database have been included. Cross-validation was based on assessment of convergent and discriminant validity. Optimal cut-offs were determined against external criteria by calculating the respective 25th and 75th percentiles mean values of RAID. External criteria included definitions for remission (REM), low disease activity (LDA), moderate disease activity (MDA) and high disease activity (HDA), cut-offs of the 28-joint disease activity score-C-reactive protein (DAS28-CRP) score. Results The RAID showed a moderate degree of correlation with respect to DAS28-CRP (rho = 0.417 ; P < 0.0001). The receiver operating characteristic (ROC) curves to discriminate the ability of RAID to distinguish patients with active and non-active disease was very good with an area under the curve (AUC) of 0.847 (95% confidence interval [CI]: 0.816 to 0.878 ; P < 0.0001). Based on the distributions of RAID in the different disease activity groups, we propose the following cut-off values for REM: RAID ≤3 ; for LDA: RAID >3 and ≤4 ; for MDA: RAID >4 and ≤6 ; for HDA: RAID >6. Mean RAID differed significantly between patients classified as REM, LDA, MDA or HDA (P = 0.001). Conclusions The cut-offs revealed good measurement characteristics in cross- validation analysis, had great discriminatory performance in distinguishing patients with different levels of disease activity and are suited for widespread use in everyday practice application and research.

Alterations in macrophage polarisation are recognised among the possible immune system abnormalities contributing to systemic sclerosis (SSc) pathogenesis.1 Macrophages have been classified as classically (M1) or alternatively (M2) activated, although growing evidence indicates that they may exhibit characteristics shared by more than one of the described phenotypes.2–5 An M2 pre-eminent phenotype has been postulated for SSc monocytes/macrophages.2 The aim of the study was to widen a phenotype characterisation (M1, M2 and mixed M1/M2) of circulating monocytes/macrophages in patients with SSc and healthy subjects (HSs) through flow cytometry analysis. Fifty-eight consecutive patients with SSc (38 limited and 20 diffuse SSc) and 27 age-matched and gender-matched HSs were enrolled after signing informed consent. SSc diagnosis was based on the 2013 American College of Rheumatology/European League Against Rheumatism criteria (online supplementary file 1).6 For flow cytometry analysis, peripheral blood was collected and anti-CD14 and anti-CD45 antibodies were used to identify the monocyte/macrophage lineage; macrophage scavenger receptors (CD204, CD163) and mannose receptor 1 (CD206) were used as M2 phenotype markers; and co-stimulatory molecules (CD80, CD86) and Toll-like receptors (TLR4, TLR2) were used as M1 phenotype markers. CD66b was used to distinguish granulocytes (Miltenyi Biotech, Germany). Flow c ytometry analysis was performed using the Navios flow cytometer and Kaluza analysis software (Beckman Coulter). A total of 5 × 10 6 cells were evaluated and more than 30 events were detected in the smallest subset investigated, according to the consensus guidelines for minimal residual disease. Results were expressed in percentages over total circulating leu c ocytes, unless otherwise specified. The non-parametric al Mann-Whitney U test was used for statistical analysis and any p value lower than 0.05 was considered statistically significant. Two initial gating strategies were used to study circulating M2-like monocytes/macrophages, the first gated CD14+cells and the second gated CD204 …

Background: Systemic sclerosis (SSc) is a disorder characterized by immune system alterations, vasculopathy and fibrosis. SSc-related interstitial lung disease (ILD) represents a common and early complication, being the leading cause of mortality. Monocytes/macrophages seem to have a key role in SSc-related ILD. Interestingly, the classically (M1) and alternatively (M2) activated monocyte/macrophage phenotype categorization is currently under revision. Our aim was to evaluate if circulating monocyte/macrophage phenotype could be used as biomarker for lung involvement in SSc. To this purpose we developed a wide phenotype characterization of circulating monocyte/macrophage subsets in SSc patients and we evaluated possible relations with lung involvement parameter values. Methods: A single centre cross-sectional study was performed in fifty-five consecutive SSc patients, during the year 2017. All clinical and instrumental tests requested for SSc follow up and in particular, lung computed tomography (CT) scan, pulmonary function tests (PFTs), Doppler echocardiography with systolic pulmonary artery pressure (sPAP) measurement, blood pro-hormone of brain natriuretic peptide (pro-BNP) evaluation, were performed in each patient in a maximum one-month period. Flow cytometry characterization of circulating cells belonging to the monocyte/macrophage lineage was performed using specific M1 (CD80, CD86, TLR2 and TLR4) and M2 surface markers (CD204, CD163 and CD206). Non-parametric tests were used for statistical analysis. Results: A higher percentage of circulating CD204(+)CD163(+)CD206(+)TLR4(+)CD80(+)CD86(+) and CD14(+)CD206(+)CD163(+)CD204(+)TLR4(+)CD80(+)CD86(+) mixed M1/M2 monocyte/macrophage subsets, was identified to characterize patients affected by SSc-related ILD and higher systolic pulmonary artery pressure. Mixed M1/M2 monocyte/macrophage subset showed higher percentages in patients positive for anti-topoisomerase antibody, a known lung involvement predictor. Conclusions: The present study shows for the first time, through a wide flow cytometry surface marker analysis, that higher circulating mixed M1/M2 monocyte/macrophage cell percentages are associated with ILD, sPAP and anti-topoisomerase antibody positivity in SSc, opening the path for research on their possible role as pathogenic or biomarker elements for SSc lung involvement.

Background Systemic sclerosis (scleroderma, SSc) is an autoimmune connective tissue disease characterized by skin and internal organ fibrosis, coupled with widespread vascular pathology. Skeletal muscle involvement in SSc has often been considered as a minor component of the disease associated with disuse. Objectives The goal of this study is to identify specific histopathological hallmarks of skeletal muscle involvement in SSc. Methods A total of 50 SSc patients presenting clinical, serological and electromyographic (EMG) features of muscle weakness, were enrolled. Patients underwent vastus lateralis biopsy, assessed for individual pathologic features including fibrosis [type I collagen (Coll-I), transforming growth factor β (TGF-β)], microangiopathy [cluster of differentiation 31 (CD31), pro-angiogenic vascular endothelial growth factor A (VEGF-A), anti-angiogenic VEGF-A165b], immune/ inflammatory response [CD4, CD8, CD20, human leucocyte antigens ABC (HLA-ABC)], and membranolytic attack complex (MAC). SSc biopsies were compared to biopsies of (n =50) idiopathic inflammatory myopathies (IIMs) and to (n =50) noninflammatory myopathies (NIMs). Ultrastructural abnormalities of SSc myopathy were also analyzed by transmission electron microscopy (TEM). Results Fibrosis in SSc myopathy (90%) is higher compared to IIM (30%, p Conclusions The predominat features of SSc-related myopathy are fibrosis, microangiopathy and humoral immunity. However, it is difficult to identify specific histopathological hallmarks of muscle involvement in SSc, since they could be present also in other (IIM/NIM) myopathies. Disclosure of Interest None declared

Background In physiological and pathological conditions, such as in tissue repair and in fibrosing diseases (i.e. systemic sclerosis, SSc), the importance of circulating fibrocytes relies on the capacity of such cells to migrate into scleroderma-affected tissues (through CXCR4/CXCL12 interaction) and to differentiate into fibroblasts/myofibroblasts, inducing fibrosis (through matrix protein deposition) (or both) [1–3]. In addition, fibrocytes seem to exert immunomodulatory effects, expressing class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR) [4]. Objectives To isolate fibrocytes from Circulating Progenitor Cells (CPCs - peripheral blood mononuclear cells PBMCs) of SSc patients and healthy subjects and to identify them by fluorescence-activated cell sorter analysis (FACS), using their specific markers: the leukocyte common antigen CD45, collagen I (COL I), the chemokine receptor CXCR4 and HLA-DR [2]. Methods Samples were collected, at basal time (t0), from 11 SSc patients, affected by SSc (treated only with different vasodilator drugs) and 5 healthy subjects (CNTs). PBMCs, isolated from 9 among the SSc patients, were cultured on fibronectin-coated plates [5]. The non-adherent cells were removed and after 8 days (t8) of culture (standardized time), the adherent spindle shaped cells were lifted through incubation in 0.05% EDTA (ice-cold). Fibrocyte identification (at both t0, t8), was performed by FACS, using anti-CD45, anti-COL I, anti-CXCR4 and anti-HLA-DR monoclonal antibodies. Results FACS analysis revealed that, at basal time (t0), among the CD45+ cells, the percentage of fibrocytes, identified as triple positive (CD45+, COL I+, CXCR4+) was 1.0±1.2% in SSc patients and 0.4±0.3% in healthy subjects (CNTs). In addition, the HLA-DR expression on fibrocytes in both SSc patients and CNTs showed low values (22.1±21.1% and 7.1±6.1%, respectively). After 8 days (t8) of culture, fibrocytes presented adherent and spindle shaped morphology. Interestingly, the FACS analysis at t8 of culture, demonstrated that the percentage of SSc fibrocytes CD45+, COL I+, CXCR4+ increased up to 52.8±27.1%, compared to basal time (t0), as well as strongly increasing the HLA-DR+ expression (90.1±22.7%). Conclusions Fibrocytes isolated from CPCs of SSc patients were confirmed to express CD45, COL I and CXCR4 molecules, but in very low percentage at the beginning. Already after 8 days of culture in proper conditions, the percentage of differentiated fibrocytes (CD45+, COL I+ and CXCR4+) from SSc patients, increased up to 50 times and the HLA-DR expression increased up to 68%. Additional markers of progressive fibrocyte differentiation are now under test to further characterize the fibrocytes phenotype(s) of SSc patients vs healthy controls. References Chesney J et al. Proc. Natl. Acad. Sci. 1997; 94:6307–6312. Bucala R. Mol Med 2015;21:S3–S5. Brunasso A.M. et al. F1000Research 2016;5:723. Blakaj and Bucala. Fibrogenesis & Tissue Repair 2012;5:S6. Pilling D et al. J Immunol Methods 2009; 351:62–70. Disclosure of Interest None declared

Background The alteration of microvascular endothelial cell (EC) functions and the presence of macrophages in the immune inflammatory infiltrate, followed by the transition of these cell types into a profibrotic phenotype, represent early and crucial pathological features of the fibrotic process in systemic sclerosis (SSc) (1). The alternatively activated macrophage subset M2a was found in several diseases characterized by extensive fibrosis (1). M2a macrophages express specific phenotype markers, CD206 (mannose receptor), CD204 and CD163 (scavenger receptors) as well as profibrotic molecules, primarily transforming growth factor-β1 (TGFβ1) (1). Endothelin-1 (ET1) and/or TGFβ1 are known to induce the transition of fibroblasts into profibrotic myofibroblasts, which are key mediators of fibrosis in SSc. Objectives To investigate the effects of ET1 in inducing a profibrotic phenotype in cultured human microvascular ECs (HMVECs) and macrophages. Methods HMVECs, at 3rd culture passage, were grown in endothelial cell medium (EGM2MV) and treated for 6 days with ET1 (100nM) or treated for 1 hr with ET1 receptor antagonist (ETA/BRA, bosentan 10μM) before stimulation with ET1. Human monocytes were isolated from peripheral blood mononuclear cells of healthy subjects using a monocyte isolation kit. The cells were maintained in RPMI growth medium for 24 hrs and then treated for 6 days with ET1 or treated for 1 hr with bosentan before stimulation with ET1. Cultured HMVECs and monocytes maintained in EGM2MV and RPMI growth medium, respectively, were used as untreated cells. Gene and protein expression of profibrotic myofibroblast markers –α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), type 1 collagen (COL1) and fibronectin (FN) – were evaluated by quantitative real time polymerase chain reaction (qRT-PCR), Western blotting (WB) and immunocytochemistry (ICC) in cultured HMVECs. Gene and protein expression of M2a phenotype markers (CD206, CD204, CD163) and TGFβ1 were investigated by qRT-PCR and WB in cultured human macrophages. Statistical analysis was carried out by Mann-Whitney non-parametric test. Results In cultured HMVECs, ET1 induced the significant upregulation of the gene expression of α-SMA, S100A4 (myofibroblast markers), COL1 and FN, compared to untreated cells (p In cultured human macrophages, ET1 induced the significant overexpression of M2a markers (p Conclusions ET1 seems to be involved in the early phases of the fibrotic process by inducing the transition of both cultured HMVECs and macrophages into a profibrotic phenotype, myofibroblast and M2a respectively (observed in SSc), a process which is apparently contrasted by ETA/BRA treatment. References Wynn TA et al. Immunity. 2016;44:450–62. Disclosure of Interest S. Soldano: None declared, P. Montagna: None declared, R. Brizzolara: None declared, A. Trombetta: None declared, A. Sulli: None declared, C. Pizzorni: None declared, M. Ghio: None declared, S. Paolino: None declared, V. Smith: None declared, M. Cutolo Grant/research support from: Actelion

Background Vitamin D (25(OH)D) deficiency seems a distinct risk factor influencing prevalence and severity of several autoimmune diseases. Several studies suggest that low serum concentrations of vitamin D are frequent in rheumatoid arthritis (RA) patients, and an inverse relationships have been reported between 25(OH)D serum concentrations and disease activity or disability. Objectives European multicentre cross-sectional study to collect data on vitamin D serum concentrations in RA patients from different countries, and to investigate the relationship with disease activity, disability and quality of life in a large population. Methods The survey involved 625 RA patients (mean age 55±11 years, mean disease duration 11±9 years) and 276 age and sex-matched healthy subjects from 13 European countries. Serum samples for 25(OH)D measurement were collected during winter time (December-March) and analyzed in a central laboratory using chemiluminescence immunoassay (DiaSorin). Thirty-six percent of RA patients were treated with vitamin D analogues. Patient past medical history, Rheumatoid Arthritis Impact Diseases (RAID) score, Health Assessment Questionnaire (HAQ) and DAS28-CRP were also collected. Statistical analysis was performed by non parametric tests. Results Mean serum concentration of 25(OH)D was found significantly lower in RA patients (17.6±9.7 ng/ml) when compared to matched controls (18.9±9.4 ng/ml) (p=0.01), Several statistically significant differences between European countries were observed (possibly linked to different latitude, sun exsposure and dietary habits) (see figure). Vitamin D deficiency ( 30 ng/ml). Male and female RA patients showed similar 25(OH)D values. Negative statistically significant correlations were found between 25(OH)D serum concentrations and RAID (p=0.05) HAQ (p=0.04) and DAS28-CRP (p Conclusions This European survey add new evidences that vitamin D insufficiency/deficiency is frequent in RA patients with statistically significant differences between several countries. Vitamin D serum concentrations negatively correlate with the clinimetric indexes for disease activity, disability and quality of life in the present cohort of RA European patients. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is characterized by early vascular abnormalities and subsequent fibroblast activation and differentiation into myofibroblasts, leading to fibrosis. Recently, endothelial-to-mesenchymal transition (EndoMT), a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, has been reported in SSc. Objectives The goal of the study was to evaluate the potential of endothelin-1 (ET-1) dual receptor antagonists bosentan (BOS) and macitentan (MAC) to antagonize EndoMT in vitro. Methods 20 patients with limited SSc were enrolled and underwent double skin biopsy (affected and nonaffected skin). Fibroblasts and microvascular endothelial cells (MVECs) were isolated from biopsies. Mono- or coculture of MVECs (isolated from nonaffected skin) with fibroblasts (isolated from affected skin and stimulated with ET-1 and transforming growth factor beta [TGF-β]) were performed. In cocultures, the MVEC layer was left undisturbed or was preincubated with either BOS or MAC. After 48 h of coculture, MVECs were analyzed for their capillary formation ability and for messenger RNA and protein expression of different vascular (CD31, vascular endothelial growth factor-A [VEGF-A], VEGF-A165b) and profibrotic (alpha-smooth muscle actin [α-SMA], collagen type I [Col I], TGF-β) molecules. Results MVECs showed a reduced capillary formation ability when cocultured with SSc fibroblasts with respect to mono cultures. CD31 and VEGF-A resulted in downregulation, while VEGF-A165b, the antiangiogenic isoform, resulted in upregulation. At the same time, mesenchymal markers α-SMA, Col I, and TGF-β resulted in overexpression in MVECs. Capillary formation ability was restored when MVECs were preincubated with BOS or MAC, also reducing the expression of mesenchymal markers and restoring CD31 expression as well as the imbalance between VEGF-A and VEGF-A165b. Conclusions BOS and MAC seem able to antagonize EndoMT phenomenon in MVECs in vitro. Blocking EndoMT is important for two reasons: first, because capillary formation ability in MVECs can be restored; second, because the endothelium-derived fibrotic development in SSc can be counteracted. Acknowledgements Thanks to Prof. Bashar Kahaleh and to Dr. Yongqing Wang from University of Toledo Medical Center, Division of Rheumatology and Immunology, OH, USA, for providing SSc microvascular endothelial cells. Disclosure of Interest None declared