BackgroundLong noncoding RNAs (LncRNAs) are a novel class of noncoding transcripts with diverse regulatory functions, e.g. imprinting regulation, dosage compensation, cell cycle regulation, pluripotency and retrotransposon silencing. Dysregulation of lncRNAs is increasingly recognized to contribute to the disease pathogenesis, such as cancer, autoimmune disorders and neurodegeneration.ObjectivesTo identify candidate lncRNAs in systemic sclerosis (SSc) and investigate their function, in particular in relation to the TGFβ pathway and myofibroblast phenotype development.MethodsRNA Sequencing Ilumina HiSeq2000 was performed in healthy and SSc skin biopsies. Human skin fibroblasts were isolated from biopsies of SSc patients and healthy controls (HC), human pulmonary smooth muscle cells were purchased from Lonza. The cells were treated with 10 ng/ml TGFβ. TGFβR1 inhibitors (SD208 and SB431542) and siRNA against SMAD3 were used to investigate TGFβ driven gene expression. The lncRNA MIR503HG was silenced in skin fibroblasts using locked nucleic acid antisense oligonucleotides (LNA GapmeRs), followed by qPCR analyses and immunofluorescence staining. The expression of MIR503HG was measured in tissue samples of liver and lung fibrosis.ResultsRNA sequencing showed a significant upregulation of the lncRNA MIR503HG (H19X) in SSc versus HC skin biopsies. Importantly, the upregulation of MIR503HG was not limited to SSc skin, but present also in the tissues from liver and lung fibrosis, indicating a broader role of MIR503HG in fibrotic diseases. While there was no difference in the basal expression of MIR503HG between SSc and HC cultured dermal fibroblasts, MIR503HG was strongly and consistently induced by TGFβ. Induction of MIR503HG by TGFβ was not limited to skin fibroblasts, but evident also in other cell types relevant for SSc, e.g. pulmonary vascular smooth muscle cells. Time curve analysis revealed that the upregulation of MIR503HG by TGFβ was strongest after 6h reaching 12.8±0.7 induction; and dose curve analysis showed a steady increase of MIR503HG over physiologically relevant TGFβ concentrations. These effects were TGFβR1 dependent as shown by the inhibition experiments with the chemical inhibitors SD208 and SB431542. Moreover, the upregulation of MIR503HG by TGFβ was significantly impaired by silencing of SMAD3, further pointing to an important role of the canonical TGFβ pathway in MIR503HG expression. The knockdown of MIR503HG in skin fibroblasts led to a strong down regulation of COL1A1, fibronectin and αSMA mRNAs, indicating a potential involvement of MIR503HG in the development of a myofibroblast phenotype. Additionally, immunofluorescence staining showed reduced αSMA formation in MIR503HG silenced skin fibroblasts.ConclusionsThis is the first study reporting changes in long-non coding RNAs in SSc and across fibrotic organs. It opens new perspectives in the pathogenesis of fibrotic diseases by this novel class of regulatory, non-coding RNAs.Disclosure of InterestE. Pachera: None declared, S. Assassi: None declared, ...

Online teilnehmen unter: www.springermedizin.de/cme Für diese Fortbildungseinheit werden 3 Punkte vergeben.

BackgroundSystemic sclerosis (SSc) is a complex autoimmune disease with extensive fibrosis of the skin and internal organs in which extracellular matrix (ECM) remodeling is a key pathogenic process. Imbalance in the formation and degradation of collagens results in fibrosis. Quantifying the tissue turnover in a highly fibrotic disease such as SSc is very important for the prediction of disease progression and therapeutic efficacy. Given the clinical heterogeneity of SSc patients, biomarkers facilitating personalized medicine approaches are highly needed.ObjectivesTo evaluate the potential of selected ECM neo-epitopes as serological biomarkers for diagnosis, prediction of clinical outcomes and disease progression in SSc.MethodsHealthy controls (HC; n=29), stable SSc (n=149), and progressive SSc patients (n=23, progression defined either as 10% decrease in FVC% predicted or increase in mRSS ≥25% and 5 points on one year clinical follow up), meeting the 2013 ACR/EULAR classification criteria were analyzed. Longitudinal clinical assessment, data recording and sera collection were done according to EUSTAR standards. ECM-degradation (C3M, VICM, C4M2, BGM) and ECM-formation biomarkers (P1NP, P4NP7S, Pro-C3, Pro-C5, Pro-C6) were measured in serum using newly developed ELISA-based assays (Nordic Bioscience). Differences in biomarker levels were analyzed with respect to several fibrosis-related clinical outcomes. Statistical analysis was performed by Man-Whitney U, Kruskal-Wallis and Spearman tests. Biomarkers' sensitivity and specificity was examined by ROC analysis.ResultsBoth ECM-degradation and ECM-formation biomarkers differed between SSc patients and HC: The expression of C4M2, Pro-C3, BGM and C3M was significantly increased in SSc patients compared to HC (p<0.0001, AUC=0.93; p<0.0001, AUC=0.74; p=0.003, AUC=0.67; p<0.0001; AUC=0.94, respectively), whereas P1NP was significantly lower (p<0.0001, AUC=0.78), Figure 1. Furthermore, Pro-C3, VICM and Pro-C6 levels were significantly higher in SSc progressors vs. HC (p<0.0001, AUC=0.86; p=0.003, AUC=0.75; p=0.0005, AUC=0.81, respectively).Currently, there is no fully validated biomarker predicting worsening of fibrosis. In this regard, most interestingly, the ECM-degradation markers C4M2, BGM, C3M were significantly lower in SSc patients showing progression of fibrosis on follow up versus SSc patients being stable on follow up (p<0.0001; p<0.008; p<0.0001, respectively). Consistently, the formation marker Pro-C6 was significantly increased (p=0.001, AUC=0.71) indicating a profound imbalance of ECM turnover in progressors. The strongest difference between progressive and stable SSc patients was seen for the ratio between the formation and degradation biomarkers Pro-C3/C3M which showed an AUC of 0.86 in progressive vs. stable SSc patients (p<0.0001).ConclusionsThese data support ECM neo-epitopes as potential new biomarkers of prognostic interest in SSc. This could help identifying patients at risk of progression of their fibrotic disease at one year follow up. The significant...

BackgroundOne of the major causes of death in systemic sclerosis (SSc) patients is heart involvement which phenotypically resembles inflammatory dilated cardiomyopathy (iDCM) [1]. Recent investigations in animal models of iDCM have demonstrated that the main source of pathological myofibroblasts originates from the bone marrow [2].ObjectivesWe hypothesized that the monocyte/macrophage compartment is responsible for the onset and progression of myocardial fibrosis in SSc. Therefore, we studied the differentiation potential of this cellular compartment and their interaction with stromal and lymphoid cell compartments.MethodsWe isolated CD14+ monocytes from the peripheral blood of SSc patients and healthy subjects and differentiated them towards a myofibroblast phenotype by stimulation with TGF-β1, IL-1β, IL-4, IL-10 and IL-13. After 7 days, cells were screened for expression of the myofibroblast marker genes α-smooth muscle actin (αSMA), fibronectin and collagen I, and Fra-2 and Wnt gene expression. Additionally, we established a coculture system allowing direct coculture of monocytes with human fibroblasts or T-cells in order to analyse the role of direct and indirect cell-to-cell-interaction on both monocyte and fibroblast differentiation.ResultsCD14+ monocytes differentiated towards a myofibroblast phenoypte as demonstrated by upregulation of αSMA (9.6 fold), fibronectin (380 fold) and collagen I (32.5 fold). This process involves Fra-2 signaling (2.2 fold upregulation) and Wnt signaling (Wnt-1 and Wnt-5a only detectable in stimulated cells). Inhibitor studies demonstrated that canonical TGF-β1 signaling is crucial for differentiation, although the application of TGF-β1 alone is not sufficient to induce in vitro differentiation in monoculture. However, direct coculture of monocytes with fibroblasts induced upregulation of αSMA (9.6 fold), fibronectin (150 fold) and collagen I (900 fold) in the presence of TGF-β1. In direct coculture with stimulated T-cells, monocytes also upregulated myofibroblast marker genes even without the addition of cytokines.ConclusionsWe showed that monocytes from the peripheral blood have the potential to differentiate towards a myofibroblast phenotype and therefore represent a potential cellular source for pathological myofibroblasts in SSc. In vivo, this differentiation might be driven by the interaction with stimulated T-cells and resident fibroblasts. A deeper insight into the pathways that drive this process might present a novel strategy to develop disease modifying treatment for SSc in general and in heart involvement in particular.ReferencesVenalis P et al., Cardiomyopathy in murine models of systemic sclerosis. Arthritis & Rheumatology. 2014 [epub ahead of print]Kania G et al., Heart-infiltrating prominin-1+/CD133+ progenitor cells represent the cellular source of transforming growth factor beta-mediated cardiac fibrosis in experimental autoimmune myocarditis. Circulation research. 2009;105(5):462-70.Disclosure of InterestV. Haunerdinger: None declared, E. Pachera: None declared...

BackgroundThere is a high unmet need for disease-modifying antifibrotic therapies in diffuse cutaneous systemic sclerosis (dcSSc) which could improve the outcome of this severe disease. Patient reported outcome measures (PROMs) are important and mandatory for randomized clinical trials (RCTs). The EULAR-endorsed ScleroID is the first comprehensive PROM specifically developed by SSc patients and experts to reflect the disease impact of SSc and showed a good performance in the clinical validation study [1]. However, most RCTs focus on dcSSc patients, hence a validated PROM to reflect the disease burden experienced by patients with dcSSc is needed and a detailed analysis of ScleroID in this subset of patients is lacking.ObjectivesTo investigate the performance of the EULAR ScleroID in patients with dcSSc as a prerequisite for its use as a PROM in RCTs testing potentially disease-modifying drugs.MethodsThis is a subanalysis of all patients with dcSSc from the large, multicentric, ScleroID validation cohort [1]. As comparators, SSc-HAQ, EQ-5D, SF-36 were included. The study had a longitudinal arm with a reliability visit at 7+/- 3 days and a 12-month follow-up visit [1]. The performance of ScleroID in dcSSc was assessed according to the OMERACT filter [1].Results152 dcSSc patients with a baseline visit were analyzed (44, 28.9% male, median age 54 years, median disease duration 7 years). ScleroID performed well as a PROM reflecting the disease impact of dcSSc: it showed a good construct validity with high Spearman’s correlation coefficients with comparators (SSc-HAQ, 0.79, 95%CI [0.69, 0.86]; HAQ-DI, 0.72 95%CI [0.60, 0.80]; SF-36 physical score, -0.69 95%CI [-0.77, -0.60]). Furthermore, the internal consistency was strong: Cronbach’s alpha was 0.87 and the split half reliability coefficient was 0.88. In the longitudinal arm, 44 patients had a test-retest reliability visit and 113 a follow-up visit, of whom 19/113 (16.8%) reported a significant change at 12 months follow-up (11 improved, 8 worsened). ScleroID showed a good consistency and discrimination ability with excellent test-retest reliability (intra-class correlation coefficient 0.89, 95%CI [0.84, 0.92]) and moderate sensitivity to change (standardized response mean 0.54), superior to the comparators (SSc-HAQ, 0.01; HAQ-DI, -0.07; SF-36 physical score, -0.43). The results are summarized in Figure 1.ConclusionThe EULAR ScleroID, which is a novel, brief, disease specific, patient-derived, disease impact PROM, performs well for patients with dcSSc. This supports the inclusion and further analysis of ScleroID as a PROM to reflect patients’ perspective in RCTs.Reference[1]Becker M, Dobrota R, Garaiman A, et al. Development and validation of a patient-reported outcome measure for systemic sclerosis: the EULAR Systemic Sclerosis Impact of Disease (ScleroID) questionnaire. Ann Rheum Dis. 2022 Apr;81(4):507-515.Figure 1.Performance of ScleroID in dcSSc by the OMERACT filter for truth and discrimination.AcknowledgementsWe sincerely thank the patients’ representatives who contributed to the ScleroID study, without whom this project would not have been possible.Disclosure of InterestsRucsandra Dobrota Speakers bureau: Actelion, Consultant of: Boehringer-Ingelheim, Grant/research support from: Pfizer, Actelion, Alexandru Garaiman: None declared, Kim Fligelstone: None declared, Ann Kennedy: None declared, Annelise Roennow: None declared, Yannick Allanore Consultant of: AbbVie, AstraZeneca, Bayer, Boehringer-Ingelheim, Mylan, Janssen, Medsenic, Prometheus, Sanofi, Roche, Grant/research support from: Alpine Immunosciences, Medsenic, OSE Immunotherapeutics, Patricia Carreira: None declared, László Czirják: None declared, Christopher P Denton: None declared, Roger Hesselstrand: None declared, Gunnel Sandqvist: None declared, Otylia Kowal-Bielecka: None declared, Cosimo Bruni Speakers bureau: Eli-Lilly, Consultant of: Boehringer-Ingelheim, Grant/research support from: Gruppo Italiano Lotta alla Sclerodermia (GILS), European Scleroderma Trials and Research Group (EUSTAR), Foundation for research in Rheumatology (FOREUM), Scleroderma Clinical Trials Consortium (SCTC), AbbVie, Marco Matucci Cerinic: None declared, Carina Mihai Speakers bureau: Boehringer Ingelheim, Janssen, MED Talks Switzerland, Mepha, and PlayToKnow AG, Consultant of: Boehringer Ingelheim, Janssen, MED Talks Switzerland, Mepha, and PlayToKnow AG, Ana Maria Gheorghiu: None declared, Ulf Müller-Ladner: None declared, Joe Sexton: None declared, Tore K. Kvien: None declared, Turid Heiberg: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Mike O. Becker Speakers bureau: Mepha, Bayer, MSD, GSK, Amgen, Novartis and Vifor, Consultant of: Mepha, Bayer, MSD, GSK, Amgen, Novartis and Vifor.