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Background/Purpose: Interferons (IFNs) seem to play an important role in the pathogenesis of juvenile dermatomyositis (JDM). Our group previously reported that expression of both type I and type II IFN related genes is increased in muscle biopsies of JDM patients and correlates with histological and clinical features of the disease. The aim of this study was to investigate expression of interferon regulated genes (IRGs), as well as serum levels of two type I and type II IFN induced chemokines (CXCL9, CXCL10) and neopterin in peripheral blood of JDM patients and to assess their correlations with clinical and laboratory findings. Methods: We collected 189 blood samples from 39 JDM patients at different time points during follow-up. In 11 patients we obtained the first blood sample at time of muscle biopsy. We measured expression of type I IRGs (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1), IFNg and type II IRGs (CXCL9, CIITA, IDO1) by quantitative PCR (qPCR) and calculated a type I and type II IFN score for muscle and blood samples; serum levels of CXCL9, CXCL10 and neopterin were analyzed by ELISA. Ten healthy subjects were used as controls (HC). At each visit, the following clinical data were recorded: physician's global assessment (PGA) of disease activity VAS (Visual Analogue Scale), cutaneous VAS, Cutaneous Assessment Tool (CAT) activity score, Childhood Myositis Assessment Score (CMAS), serum levels of creatine phosphokinase (CK, IU/l), presence of myositis specific or myositis associated antibodies (MSA/MAA), prednisone (or equivalent) dose (mg/kg/daily), ongoing immunosuppressive medications. Results: Serum levels of CXCL9 where significantly correlated with muscle expression of IFNg and type II IFN score. The correlation of CXCL10 levels with muscle type I and type II IFN score was weaker. Muscle expression of CXCL9 and CXCL10 correlated with serum levels of these chemokines. Type I IFN score in blood of JDM patients was increased compared to HC and significantly correlated with PGA, cutaneous VAS, CAT activity score. Serum levels of CXCL9 and CXCL10 were significantly higher in JDM patients compared to HC. MSA positive JDM patients showed higher levels of CXCL9 and CXCL10 compared to MSA negative patients. CXCL10 levels were correlated with PGA and CMAS, but not with cutaneous disease activity. CXCL9 showed no significant association with the evaluated clinical features. Neopterin levels were significantly correlated with PGA, cutaneous VAS, CAT activity score and CMAS. Conclusion: Our findings indicate that expression of IRGs, measured as type I IFN score, and serum levels of CXCL10 and neopterin reflect specific features of disease activity in JDM, supporting their role as valuable disease biomarkers. Disclosure: R. Nicolai, None; I. Caiello, None; R. Lucilla, None; S. Rosina, None; F. Licciardi, None; L. Bracci Laudiero, None; A. Ravelli, Angelini, AbbVie, Bristol-Myers Squibb, Johnson & Johnson, Novartis, Pfizer, Reckitt Benkiser, and Roche, 2, 5, 8; F. De Benedetti, AbbVie, 2, BMS, 2, Novartis, 2, Novartis, Novimmune, Hoffmann- La Roche, SOBI, AbbVie, Pfizer, 2, Novimmune, 2, Pfizer, 2, Roche, 2, Sanofi, 2, Sobi, 2, Swedish Orphan Biovitrum, 2, UCB, 2; G. Moneta, None.

Background/Purpose: Nerve Growth Factor (NGF) production in the organism is a strictly regulated process. Inflammation increases NGF expression and indeed high NGF levels are a feature of synovial fluid and tissue in patients with chronic arthritis. Although all mononuclear cells express NGF receptors, it is, however, unclear what are the biological effects of these high NGF levels on immune cell activity. In order to evaluate the ability of mononuclear cells to respond to NGF in vivo, we analyzed the expression of TrkA and p75-NTR in mononuclear cells of patients with juvenile idiopathic arthritis (JIA). For better understanding the possible effect of NGF during the inflammatory response we studied how intracellular signalling pathways induced by NGF affect TLR downstream signalling in monocytes, which are key players in inflammation and a critical link between innate and adaptive immunity. Methods: Mononuclear cells were isolated from synovial fluid and peripheral blood samples of 25 children with JIA and age matched controls by Ficoll-Hypaque density centrifugation. Healthy donor monocytes, purified using Percoll discontinuous density gradients, were stimulated with TLR ligands with or without NGF addition. The expression of TrkA and p75NTR and the effects of TrkA activation or inhibition on TLR signalling and cytokine release were evaluated using real-time PCR, western blot and ELISA. Results: We found that mononuclear cells of JIA patients from both peripheral blood and synovial fluid are characterized by a significant decrease in TrkA expression, while p75-NTR expression remains stable. In normal monocytes, phosphorylation of TrkA after NGF binding activated intracellular pathways that interfered with TLR signalling. Indeed NGF addition increased Akt phosphorylation, inactivated GSK3 and reduced IkB phosphorylation. Blocking TrkA enhanced inflammatory cytokine production, while reducing IL-10 synthesis and resulted in a greater activation of the NF-kB pathway and in inhibition of the PI3K pathway. Conclusion: Our results imply that NGF is a relevant component of the endogenous mechanisms limiting excessive inflammatory response. Indeed, through TrkA NGF potentiates anti-inflammatory responses usually activated after TLR stimulation. The well-known increase in NGF in chronic arthritis patients may be de facto not efficacious in controlling inflammation because of the decrease of TrkA expression observed in JIA patients. This downregulation of TrkA expression may influence the balance between inflammatory and anti-inflammatory pathways.

Background/Purpose: Interferons (IFNs) seem to be important contributors in the pathogenesis of juvenile dermatomyositis (JDM). Our group previously reported that expression of both type I and type II IFNs is increased in muscle biopsies of JDM patients and correlates with histological and clinical aspects of the disease, and other authors described up-regulation of interferon regulated genes (IRGs) in blood samples of JDM patients. The aim of this study was to investigate expression of IRGs (measured as type I IFN score) and serum levels of two type II IFN (IFN?) induced chemokines (CXCL9, CXCL10) in peripheral blood of JDM patients. Furthermore, we wanted to evaluate possible correlations of type I IFN score, as well as of CXCL9 and CXCL10, with clinical and laboratory findings during disease course. Methods: We collected 79 blood samples from 26 JDM patients at different times during the disease course. We measured expression of IRGs (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) by real-time PCR and calculated a type I IFN score; serum levels of CXCL9 and CXCL10 were analyzed by ELISA. The first blood sample was collected at the same time the patient underwent muscle biopsy in six patients. At each visit, the following clinical data were recorded: physician's global assessment of disease activity VAS (Visual Analogue Scale), cutaneous VAS, Cutaneous Assessment Tool (CAT) activity and damage score, Childhood Myositis Assessment Score (CMAS), serum levels of creatine phosphokinase (CK, IU/l), prednisone (or equivalent) dose (mg/kg/daily), ongoing immunosuppressive medications. Results: Blood expression levels of IRGs were up-regulated in untreated JDM patients at diagnosis compared to healthy controls. Type I IFN score progressively reduced after start of glucocorticoid and immunosuppressive therapy. CXCL9 and CXCL10 serum levels had a similar trend. These patients also showed an increased expression of IRGs, CXCL9 and CXCL10 in muscle biopsies obtained at diagnosis. We found that the type I IFN score correlates with cutaneous disease activity assessed by the CAT activity score (p = 0.027), whereas there was no significant correlation with cutaneous VAS (p = 0.09) or CAT damage score (p = 0.88). Serum levels of CXCL10 showed correlation with serum CK levels (p = 0.042) and muscular function assessed by CMAS (p = 0.021). None of the evaluated clinical features showed correlation with serum levels of CXCL9. Conclusion: Our findings indicate that in addition to CXCL10, already suggested as potential biomarker in JDM, expression of IRGs measured as type I IFN score reflects disease activity in JDM.