Marzia Rossato

The role of genetics and epigenetics in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a complex autoimmune disease of unclear aetiology. A multitude of genetic studies, ranging from candidate-gene studies to genome-wide association studies, have identified a large number of genetic susceptibility factors for SSc and its clinical phenotypes, but the contribution of these factors to disease susceptibility is only modest. However, in an endeavour to explore how the environment might affect genetic susceptibility, epigenetic research into SSc is rapidly expanding. Orchestrated by environmental factors, epigenetic modifications can drive genetically predisposed individuals to develop autoimmunity, and are thought to represent the crossroads between the environment and genetics in SSc. Therefore, in addition to providing a comprehensive description of the current understanding of genetic susceptibility underlying SSc, this Review describes the involvement of epigenetic phenomena, including DNA methylation patterns, histone modifications and microRNAs, in SSc.

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis

Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)–mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.

Expression of miR-618 in plasmacytoid dendritic cells from systemic sclerosis patients is associated with their altered frequency and activation

Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)–mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.

Can baseline serum microRNAs predict response to TNF-alpha inhibitors in rheumatoid arthritis?

BackgroundIn rheumatoid arthritis, prediction of response to TNF-alpha inhibitor (TNFi) treatment would be of clinical value. This study aims to discover miRNAs that predict response and aims to replicate results of two previous studies addressing this topic.MethodsFrom the observational BiOCURA cohort, 40 adalimumab- (ADA) and 40 etanercept- (ETN) treated patients were selected to enter the discovery cohort and baseline serum profiling on 758 miRNAs was performed. The added value of univariately selected miRNAs (pu2009<u20090.05) over clinical parameters in prediction of response was determined by means of the area under the receiver operating characteristic curve (AUC-ROC). Validation was performed by TaqMan single qPCR assays in 40 new patients.ResultsExpression of miR-99a and miR-143 predicted response to ADA, and miR-23a and miR-197 predicted response to ETN. The addition of miRNAs increased the AUC-ROC of a model containing only clinical parameters for ADA (0.75 to 0.97) and ETN (0.68 to 0.78). In validation, none of the selected miRNAs significantly predicted response. miR-23a was the only overlapping miRNA compared to the two previous studies, however inversely related with response in one of these studies. The reasons for the inability to replicate previously proposed miRNAs predicting response to TNFi and replicate those from the discovery cohort were investigated and discussed.ConclusionsTo date, no miRNA consistently predicting response to TNFi therapy in RA has been identified. Future studies on this topic should meet a minimum of standards in design that are addressed in this study, in order to increase the reproducibility.

Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis

OBJECTIVEnMicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease.nnnMETHODSnThe levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (nu202f=u202f22), systemic lupus erythematosus (SLE) (nu202f=u202f33) and primary Sjögrens syndrome (pSS) (nu202f=u202f23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells.nnnRESULTSn30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes.nnnCONCLUSIONSnOur findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

B cell depleting therapy regulates splenic and circulating T follicular helper cells in immune thrombocytopenia

B cells are involved in immune thrombocytopenia (ITP) pathophysiology by producing antiplatelet auto-antibodies. However more than a half of ITP patients do not respond to B cell depletion induced by rituximab (RTX). The persistence of splenic T follicular helper cells (TFH) that we demonstrated to be expanded during ITP and to support B cell differentiation and antiplatelet antibody-production may participate to RTX inefficiency. Whereas it is well established that the survival of TFH depends on B cells in animal models, nothing is known in humans yet. To determine the effect of B cell depletion on human TFH, we quantified B cells and TFH in the spleen and in the blood from ITP patients treated or not with RTX. We showed that B cell depletion led to a dramatic decrease in splenic TFH and in CXCL13 and IL-21, two cytokines predominantly produced by TFH. The absolute count of circulating TFH and serum CXCL13 also decreased after RTX treatment, whatever the therapeutic response. Therefore, we showed that the maintenance of TFH required B cells and that TFH are not involved in the inefficiency of RTX in ITP.

Patients with gout have short telomeres compared with healthy participants: association of telomere length with flare frequency and cardiovascular disease in gout

Aim and background Chronic inflammation associates with increased senescence, which is a strong predictor for cardiovascular disease. We hypothesised that inflammation accelerates senescence and thereby enhances the risk of cardiovascular disease in gout. Methods We assessed replicative senescence by quantifying telomere length (TL) in a discovery cohort of 145 Dutch patients with gout and 273 healthy individuals and validated our results in 474 patients with gout and 293 healthy participants from New Zealand. Subsequently, we investigated the effect of cardiovascular disease on TL of all participants. Also, we measured TL of CD4+ and CD8+ T lymphocytes, B lymphocytes, monocytes, natural killer cells and plasmacytoid dendritic cells. Additionally, we assessed the potential temporal difference in TL and telomerase activity. Results TL in PBMCs of healthy donors decreased over time, reflecting normal ageing. Patients with gout demonstrated shorter telomeres (p=0.001, R2=0.01873). In fact, the extent of telomere erosion in patients with gout was higher at any age compared with healthy counterparts at any age (p<0.0001, R2=0.02847). Patients with gout with cardiovascular disease had the shortest telomeres and TL was an independent risk factor for cardiovascular disease in patients with gout (p=0.001). TL was inversely associated with the number of gouty flares (p=0.005). Conclusions Patients with gout have shorter telomeres than healthy participants, reflecting increased cellular senescence. Telomere shortening was associated with the number of flares and with cardiovascular disease in people with gout.

Earliest Phase of Systemic Sclerosis Typified by Increased Levels of Inflammatory Proteins in the Serum

Patients with definite systemic sclerosis (SSc) who lack fibrotic features can be stratified into an intermediate stage of disease severity between preclinical/early SSc (EaSSc) and fibrotic subsets (limited cutaneous SSc [lcSSc] and diffuse cutaneous SSc [dcSSc]). The aim of the present study was to molecularly characterize nonfibrotic SSc and EaSSc on the basis of a broad panel of serum markers of inflammation and tissue damage, in order to increase the knowledge of the pathophysiologic mechanisms underlying SSc progression before the development of fibrosis.

A Disease-Associated MicroRNA Cluster Links Inflammatory Pathways and an Altered Composition of Leukocyte Subsets to Noninfectious Uveitis

PurposenThe cause of noninfectious uveitis (NIU) is poorly understood but is considered to be mediated by a complex interplay between genetic, environmental, and-relatively unexplored-epigenetic factors. MicroRNAs (miRNAs) are noncoding small RNAs that are important epigenetic regulators implicated in pathologic signaling. Therefore, we mapped the circulating miRNA-ome of NIU patients and studied miRNA perturbations within the broader context of the immune system.nnnMethodsnWe designed a strategy to robustly identify changes in the miRNA profiles of two independent cohorts totaling 54 untreated patients with active and eye-restricted disease and 26 age-matched controls. High-resolution miRNA-ome data were obtained by TaqMan OpenArray technology and subsequent RT-qPCR. Flow cytometry data, and proteomic data spanning the cellular immune system, were used to map the uveitis-miRNA signature to changes in the composition of specific leukocyte subsets in blood.nnnResultsnUsing stringent selection criteria, we identified and independently validated an miRNA cluster that is associated with NIU. Pathway enrichment analysis for genes targeted by this cluster revealed significant enrichment for the PI3K/Akt, MAPK, FOXO, and VEGF signaling pathways, and photoreceptor development. In addition, unsupervised multidomain analyses linked the presence of the uveitis-associated miRNA cluster to a different composition of leukocyte subsets, more specifically, CD16+CD11c+HLA-DR- cells.nnnConclusionsnTogether, this study identified a unique miRNA cluster associated with NIU that was related to changes in leukocyte subsets demonstrating systemic changes in epigenetic regulation underlying NIU.

THU0268 Decreased Expression of MIR-130A and MIR-708 in Type-1 Classical Dendritic Cells of Sjögren's Syndrome Patients Indicates Their Dysregulation

Background Primary Sjögrens syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the salivary and lachrymal exocrine glands that leads to dryness of mouth and eyes. Type-1 classical dendritic cells (cDCs) are very potent antigen presenting cells known to induce strong T-cell proliferation and cytokine production. Despite the fact that especially cDC1s are candidate key players in the activation of local T and B-cells in pSS, they have rarely been studied in pSS. Objectives Considering the critical role of micro-RNAs (miRNAs) in regulation of gene expression, we investigated miRNA expression in isolated cDC1s of pSS patients. Methods Two independent cohorts (discovery and validation) were established including a total of 29 pSS patients. 17 healthy controls (HC) were included as control group. cD1c+CD19- cells were isolated from peripheral blood using MACS and we performed miRNA profiling of 758 miRNA targets using the OpenArray platform in the donors included in the discovery cohort. A selection of the miRNAs found to be differentially expressed in pSS versus HC was measured in the validation cohort. We performed pathway enrichment with the experimentally supported targets of the validated miRNAs. Results A total of 24 miRNAs were downregulated in pSS patients versus HC in the discovery cohort (all at least p<0.05 and 2log expression difference). Of these, 16 targets were selected to be validated in validation cohort. Two miRNAs, miR-130a and miR-708, were significantly downregulated in pSS patients in both cohorts (p<0.05). Pathway enrichment showed that the experimentally supported targets of these miRNAs are mainly involved in vesicle trafficking and a number of growth-factor signalling pathways (p<0.05, FDR corrected), including epidermal growth factor (EGF) signalling and endocytosis. We are currently linking the miRNA expression data to RNAseq data and performing functional experiments with primary DCs to dissect the effects of miRNA dysregulation in these cells. Conclusions We here for the first time show differentially expressed miRNAs in an isolated immune cell subset of pSS patients. In addition, this is the first evidence for dysregulation of primary peripheral blood type-1 cDCs in pSS. Considering threimportant role of miRNAs in cell function, this suggests that targeting aberrant miRNA expression may provide tools to modulate cDC activity. Disclosure of Interest None declared