V. Liakouli

Early assessment of sub-clinical cardiac involvement in systemic sclerosis (SSc) using delayed enhancement cardiac magnetic resonance (CE-MRI)

OBJECTIVES Systemic sclerosis heart involvement (SHI) is one of systemic sclerosis (SSc) most frequent complications, both in diffuse (dcSSc) and limited (lcSSc) cutaneous forms of disease. Nowadays, SHI is a major factor decreasing SSc survival rate because, when clinically evident, is associated with 70% of mortality at 5 years. SHI shows different forms, primary and/or secondary. Primary myocardial SHI is characterized by fibrosis. Aim of our study is to assess the presence and pattern of fibrosis as detected by cardiac magnetic resonance (CMR) in systemic sclerosis. METHODS In this study, we used CE-MRI (contrast enhanced-MRI) in 58 female SSc patients. Images were evaluated to obtain functional parameters and to see presence, location and pattern (nodular, linear or diffuse) of myocardial LE, sign of fibrosis. CE-MRI findings were correlated with patients clinical setting. RESULTS Myocardial fibrosis was detected in 25 of 58 patients (43%). The main finding observed in 16 of these 25 patients was a late enhancement showing a linear pattern, without coronary distribution and sparing the sub-endocardial myocardial layers. A patchy nodular enhancement pattern was observed in 9 patients (36%). Patients with linear pattern presented dcSSc, on the contrary patients with nodular LE displayed the lcSSc form. CONCLUSIONS This study shows that CE-MRI is a reliable technique to detect SHI earlier than other methods. SHI increase passive myocardial stiffness, proportional to collagen deposition degree, leading to cardiac remodelling with possible development of heart failure, even with normal ejection fraction. An early treatment of SHI might improve SSc patients outcome.

Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy.

Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc–mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, β‐galactosidase (β‐Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co‐culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4+ cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc–MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0–G1 phase, without significant differences between SSc and HC. SSc–MSCs showed an increased positive β‐Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, β‐Gal staining increased significantly in SSc–MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc– and HC–MSCs. Interleukin (IL)‐6 and transforming growth factor (TGF)‐β‐related transcripts and their protein levels were significantly higher in SSc–MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL‐6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.

Monocytes from patients with rheumatoid arthritis and type 2 diabetes mellitus display an increased production of interleukin (IL)-1β via the nucleotide-binding domain and leucine-rich repeat containing family pyrin 3(NLRP3)-inflammasome activation: a possible implication for therapeutic decision in these patients

A better understanding about the mechanisms involved in the pathogenesis of type 2 diabetes mellitus (T2D) showed that inflammatory cytokines such as tumour necrosis factor (TNF) and interleukin (IL)‐1β play a pivotal role, mirroring data largely reported in rheumatoid arthritis (RA). IL‐1β is produced mainly by monocytes (MO), and hyperglycaemia may be able to modulate, in the cytoplasm of these cells, the assembly of a nucleotide‐binding domain and leucine‐rich repeat containing family pyrin (NLRP3)‐inflammosome, a cytosolic multi‐protein platform where the inactive pro‐IL‐1β is cleaved into active form, via caspase‐1 activity. In this paper, we evaluated the production of IL‐1 β and TNF, in peripheral blood MO of patients affected by RA or T2D or both diseases, in order to understand if an alteration of the glucose metabolism may influence their proinflammatory status. Our data showed, after 24 h of incubation with different glucose concentrations, a significantly increased production of IL‐1β and TNF in all evaluated groups when compared with healthy controls. However, a significant increase of IL‐1β secretion by T2D/RA was observed when compared with other groups. The analysis of relative mRNA expression confirmed these data. After 24 h of incubation with different concentrations of glucose, our results showed a significant increase in NLRP3 expression. In this work, an increased production of IL‐1β by MO obtained from patients affected by both RA and T2D via NLRP3‐inflammasome activation may suggest a potential IL‐1β targeted therapy in these patients.

Perivascular Cells in Diffuse Cutaneous Systemic Sclerosis Overexpress Activated ADAM12 and Are Involved in Myofibroblast Transdifferentiation and Development of Fibrosis

Objective. Microvascular damage is pivotal in the pathogenesis of systemic sclerosis (SSc), preceding fibrosis, and whose trigger is not still fully understood. Perivascular progenitor cells, with profibrotic activity and function, are identified by the expression of the isoform 12 of ADAM (ADAM12) and this molecule may be upregulated by transforming growth factor-β (TGF-β). The goal of this work was to evaluate whether pericytes in the skin of patients with diffuse cutaneous SSc (dcSSc) expressed ADAM12, suggesting their potential contribution to the fibrotic process, and whether TGF-β might modulate this molecule. Methods. After ethical approval, mesenchymal stem cells (MSC) and fibroblasts (FB) were isolated from bone marrow and skin samples collected from 20 patients with dcSSc. ADAM12 expression was investigated in the skin and in isolated MSC and FB treated with TGF-β by immunofluorescence, quantitative real-time PCR, and western blot. Further, we silenced ADAM12 expression in both dcSSc-MSC and -FB to confirm the TGF-β modulation. Results. Pericytes and FB of dcSSc skin showed an increased expression of ADAM12 when compared with healthy control skin. TGF-β in vitro treatment induced a significant increase of ADAM12 in both SSc-MSC and -FB, with the higher levels observed in dcSSc cells. After ADAM12 silencing, the TGF-β ability to upregulate α-smooth muscle actin in both SSc-MSC and SSc-FB was inhibited. Conclusion. Our results suggest that in SSc, pericytes that transdifferentiate toward activated FB are present in the vascular tree, and TGF-β, while increasing ADAM12 expression, may modulate this transdifferentiation.

The CD68+/H-ferritin+ cells colonize the lymph nodes of the patients with adult onset Still's disease and are associated with increased extracellular level of H-ferritin in the same tissue: correlation with disease severity and implication for pathogenesis

In this work, we aimed to evaluate the levels of ferritin enriched in H subunits (H‐ferritin) and ferritin enriched in L subunits (L‐ferritin) and the cells expressing these two molecules in the lymph node (LN) biopsies obtained from adult‐onset Stills disease (AOSD) patients, and the possible correlation among these data and the severity of the disease. Ten patients with AOSD underwent LN biopsy. All the samples were stained by immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H‐ferritin and L‐ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68+/H‐ferritin+ or CD68+/L‐ferritin+ cells and the clinical picture. Immunofluorescence analysis demonstrated an increased tissue H‐ferritin expression in the LNs of AOSD patients. This increased expression correlated with the severity of the disease. An increased number of CD68 macrophages expressing H‐ferritin was observed in the LN samples of our patients. Furthermore, we observed that the number of CD68+/H‐ferritin+ cells correlated significantly with the severity of the clinical picture. Our data showed an imbalance between the levels of H‐ and L‐ferritin in LNs of AOSD patients and the evidence of an increased number of CD68+/H‐ferritin+ cells in the same organs. Furthermore, a correlation among both the tissue H‐ferritin levels and the CD68+/H‐ferritin+ cells and the clinical picture was observed.

H-ferritin and CD68+/H-ferritin+ monocytes/macrophages are increased in the skin of adult-onset Still's disease patients and correlate with the multi-visceral involvement of the disease

Adult‐onset Stills disease (AOSD) patients may show an evanescent salmon‐pink erythema appearing during febrile attacks and reducing without fever. Some patients may experience this eruption for many weeks. During AOSD, exceptionally high serum levels of ferritin may be observed; it is an iron storage protein composed of 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 observed in different tissues. In this work, we aimed to investigate the skin expression of both H‐and L‐ferritin and the number of macrophages expressing these molecules from AOSD patients with persistent cutaneous lesions. We observed an increased expression of H‐ferritin in the skin, associated with an infiltrate in the biopsies obtained from persistent cutaneous lesions of AOSD patients. Furthermore, a positive correlation between H‐ferritin skin levels as well as the number of CD68+/H‐ferritin+ cells and the multi‐visceral involvement of the disease was observed. Our data showed an increased expression of H‐ferritin in the skin of AOSD patients, associated with a strong infiltrate of CD68+/H‐ferritin+ cells. Furthermore, a correlation between the levels of H‐ferritin as well as of the number of CD68+/H‐ferritin+ cells and the multi‐visceral involvement of the disease was observed.

H-ferritin and proinflammatory cytokines are increased in the bone marrow of patients affected by macrophage activation syndrome

Macrophage activation syndrome (MAS) is hyperinflammatory life‐threatening syndrome, associated typically with high levels of serum ferritin. This is an iron storage protein including heavy (H) and light (L) subunits, categorized on their molecular weight. The H‐/L subunits ratio may be different in tissues, depending on the specific tissue and pathophysiological status. In this study, we analysed the bone marrow (BM) biopsies of adult MAS patients to assess the presence of: (i) H‐ferritin and L‐ferritin; (ii) CD68+/H‐ferritin+ and CD68+/L‐ferritin+; and (iii) interleukin (IL)‐1β, tumour necrosis factor (TNF) and interferon (IFN)‐γ. We also explored possible correlations of these results with clinical data. H‐ferritin, IL‐1β, TNF and IFN‐γ were increased significantly in MAS. Furthermore, an increased number of CD68+/H‐ferritin+ cells and an infiltrate of cells co‐expressing H‐ferritin and IL‐12, suggesting an infiltrate of M1 macrophages, were observed. H‐ferritin levels and CD68+/H‐ferritin+ cells were correlated with haematological involvement of the disease, serum ferritin and C‐reactive protein. L‐ferritin and CD68+/L‐ferritin+ cells did not correlate with these parameters. In conclusion, during MAS, H‐ferritin, CD68+/H‐ferritin+ cells and proinflammatory cytokines were increased significantly in the BM inflammatory infiltrate, pointing out a possible vicious pathogenic loop. To date, H‐ferritin and CD68+/H‐ferritin+ were associated significantly with haematological involvement of the disease, suggesting biomarkers assessing severity of clinical picture.

Advances in immunopathogenesis of macrophage activation syndrome during rheumatic inflammatory diseases: toward new therapeutic targets?

ABSTRACT Introduction: Macrophage activation syndrome (MAS) is a severe, hyperinflammatory life-threatening syndrome, generally complicating different rheumatic diseases. Despite the severity of the disease, little is known about the pathogenic mechanisms and, thus, possible targeted therapies in the management of these patients. Areas covered: In this review, we aimed to update the current pathogenic knowledge of MAS, during rheumatic diseases, focusing mainly on immunologic abnormalities and on new possible therapeutic strategies. Expert commentary: The difficult pathogenic scenario of MAS, in which genetic defects, predisposing diseases, and triggers are mixed together with the high mortality rate, make it difficult to manage these patients. Although most efforts have been focused on investigating the disease in children, in recent years, several studies are trying to elucidate the possible pathogenic mechanism in adult MAS patients. In this context, genetic and immunological studies might lead to advances in the knowledge of pathogenic mechanisms and possible new therapeutic targets. In the future, the results of ongoing clinical trials are awaited in order to improve the management and, thus, the survival of these patients.

FRI0437 Decreased Expression of Angiopoetin 1 on Perivascular Mesenchymal Stem Cells from SSC Patients Induces an Anti Angiogenetic Effect, when Co-Cultured with Endothelial Cells

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

The Emerging Role of IL-1 Inhibition in Patients Affected by Rheumatoid Arthritis and Diabetes

BACKGROUND Although in the past, prevention of the joint destruction and disability was strongly emphasised in Rheumatoid Arthritis (RA), at present, a growing body of evidence is focused at identifying the best management of associated comorbidities, such as Type 2 Diabetes (T2D). Recently, the hypothesis that blocking pro-inflammatory activity may be helpful in the treatment of some comorbidities has been proposed in RA patients. OBJECTIVE We reviewed the role of IL-1β during RA and T2D, the efficacy of IL-1 blocking agents in controlling both diseases and, possible, decreasing the concomitant enhanced atherosclerotic process. METHOD After literature search, the available evidence has been selected and commented in the text. RESULTS During RA, it is well known that different inflammatory cytokines, such as interleukin-1β (IL-1β), are pivotal pathogenic mediators and their role has been largely confirmed in clinical settings. Similarly, it has been shown that the excess of nutrients, secondary to over-nutrition, may activate the immune system, leading to an increased production of inflammatory cytokines, including IL-1β, suggesting new possible therapeutic targets. CONCLUSION Although further studies are needed to fully investigate the pathogenic interplay between inflammation and metabolic disorders, IL-1β has been implicated in both RA and T2D pathogenic mechanisms. Intriguingly, the potential role of anti-IL-1 drugs has been proposed in RA patients affected by T2D.