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Interstitial lung disease (ILD) affects up to 40% of patients with systemic sclerosis (SSc) and is the leading cause of death.1 The risk of ILD is increased in SSc patients who are older and antitopoisomerase-1 antibody (ATA) positive.2 SSc-ILD shares features with idiopathic pulmonary fibrosis (IPF), although these two fibrosing lung diseases have different natural histories. Telomere shortening is the best-defined risk factor for IPF. In familial IPF, mutations in seven telomerase and telomere-related genes explain familial clustering in 30% of cases.3 Sporadic forms of IPF are also associated with telomere shortening in 50%–60% of cases.4 5 In IPF, short telomeres are found across cell types, which is consistent with an inherited and systemic defect in telomere maintenance.4 Previous studies of telomere length (TL) in patients with SSc have yielded conflicting results, with some studies showing longer and others shorter TL compared with age-matched controls.6 7 A major caveat in these studies is that TL was measured using techniques that are both highly variable and fail to distinguish TL among leucocyte subsets. Moreover, these studies failed to examine the impact of ILD on TL.We therefore sought to determine TL in patients with SSc and associated ILD using a clinically validated robust assay that allows accurate measurement of TL in lymphocytes and granulocytes by flow cytometry and fluorescence in situ hybridisation (flowFISH).8 9 Subjects were evaluated at the Northwestern Scleroderma Program between 2013 and 2014 and …

Background:Myofibroblasts are the orchestrators of aberrant extracellular matrix (ECM) remodelling in fibrosis. Actin cytoskeleton is a central hub that integrates mechanical signals to promote myofibroblast differentiation and ECM remodelling. Targeting these pathways could represent a novel antifibrotic strategy. We have recently shown that metabolic intermediate dimethyl-α-ketoglutarate (dm-αKG) blocks TGFβ-driven myofibroblast differentiation in dermal fibroblasts (DF).Objectives:To investigate the mechanisms by which dm-αKG regulates TGFβ-driven myofibroblast differentiation and inflammatory responses in DF.Methods:DF from healthy controls and patients with systemic sclerosis (SSc) were treated with TGFβ (10 ng/ml) and/or dm-αKG (6 mM) for 24h, 48h and 72h. RNA sequencing (Ilumina 2000, n=3 per experimental group) was followed by the analysis of differentially expressed genes (DeSEQ2, log2 fold ≥ |0.5|, padj< 0.01), pathway enrichment analysis (GO terms) and supervised PCA analysis (ClustVis). Protein amounts (fibronectin, αSMA, IL-6), cell contraction and apoptosis were measured with Western blot (n=6), ELISA (n=4), collagen gel contraction assay (n=4) and real time Annexin V assay (n=6). Significance (pResults:TGFβ (24h) altered the expression of 4076 genes in DF as determined by RNA-seq, among which 1864 genes were upregulated. The upregulated genes were enriched in GO biological processes/molecular functions/cellular compartments related to ECM organization (p=1e-07), Wnt signalling (p=5e-06), actin binding (p=3e-07), focal adhesion (p=1e-10), stress fibers (p=3e-07) and actin cytoskeleton (p= 3e-06). Dm-αKG altered the expression of 589 genes in TGFβ-treated DF compared to TGFβ only. The most downregulated pathways in DF treated with dm-αKG + TGFβ compared to TGFβ only included actin binding (p=5e-05), muscle contraction (p=0.001), ECM organization (p=0.008), focal adhesion (p=0.01), Z disk (p=0.01) and stress fibers (p=0.03). Specifically, dm-aKG significantly (p-0.5) decreased the expression of many TGFβ-induced genes involved in actin organization and focal adhesion (NEXN, FRMD5, ANTXR1, ACTC1, LIMCH1, SORBS2, TGM2, CSRP2, CAP2, LMO7, FZD2), muscle contraction (SNTB1, LMOD1, ANKRD1, SULF1, JPH2, CAVIN4, OXTR, DYSF, FBXO32) and ECM organization (COL10A1, COL11A1, HAPLN1, MMP14, MMP3, SPINT2, GREM1, MATN3, ADAMTS4). The PCA analysis revealed that the experimental treatment (PC1, Fig 1A) accounted for 61% variability in the expression of these genes, while 19% was attributed to interdonor variability (PC2). Dm-αKG diminished TGFβ-induced production of αSMA protein (72h, p=0.02, mean O.D. ± SD in TGFβ + dm-αKG vs. TGFβ: 0.34 ± 0.38 vs. 3.1 ± 2.3) and repressed TGFβ-driven secretion of fibronectin protein (72h, p=0.047, 0.5 ± 0.1 vs. 1.2 ± 0.6). Dm-αKG reduced the contractile capacity of TGFβ-stimulated DF in collagen gel contraction assay (p=0.003, 0 vs. 67.1 ± 5.4%). Additionally, dm-αKG decreased TGFβ-driven production of IL-6 transcripts (24h, p=0.05, 2.9 ± 0.6 vs 1.9 ± 0.3) and protein (24h, p=0.0005, 5.9 ± 1.2 vs 3 ± 0.7, Fig 1B), but did not increase the apoptosis of DF (24h, 48h, 72h).Fig 1.A Supervised PCA analysis of RNA-seq data. B. IL-6 secretion (ELISA).Conclusion:Dm-αKG counteracted TGFβ-induced myofibroblast differentiation by regulating the cytoskeleton organization and ECM dynamics in DF and blocked the TGFβ-induced IL-6 production. This closely links metabolism to inflammatory and pro-fibrotic responses in DF. Therefore, regulating intracellular αKG might offer a novel strategy in combating the inflammatory and fibrotic stages of skin fibrosis in SSc.Acknowledgments:This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology.Disclosure of Interests:Blaž Burja: None declared, Gabriela Kania: None declared, Matija Tomsic: None declared, Snežna Sodin-Šemrl: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Katja Lakota: None declared, Mojca Frank-Bertoncelj: None declared

Background Influenza is a frequent infectious disease affecting more than 20% of the world population yearly. It can cause severe complications in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis (RA). In immunocompromised patients vaccinations are especially recommended. Influenza vaccines are generally safe and effective. However, RA patients require cautiousness in terms of vaccine immunogenicity especially when treated with immunomodulatory drugs. Objectives To determine the immunogenicity of a seasonal influenza vaccine in RA patients, its timeline and the influence of different medications on patients9 immune response. Methods 137 RA patients (38 males, 99 females, median age 33) and 54 healthy controls (HC; 11 males, 43 females, median age 54) were vaccinated with trivalent seasonal influenza vaccine (Sanofi Pasteur) containing purified haemagglutinins A/Brisbane/59/2007 (H1N1), A/Brisbane/10//2007 (H3N2), B/Brisbane/60/2008 (B). Sera were collected just before vaccination (baseline), 18-90 days and >180 days after vaccination. Measurement of antibodies against surface membrane proteins was performed using haemagglutination inhibition (HI) tests, while detection of antibodies against inner viral proteins of influenza A and influenza B viruses was tested by IgG and IgA ELISA. Results Using HI test, at baseline, RA patients were seroprotected (antibody titres ≥40) against seasonal influenza viruses A(H1N1), A(H3N2) and B in 58% (79/137), 97% (133/137) and 90% (123/137) respectively, versus 74% (40/54), 100% (54/54) and 83% (45/54) in HC. At the time of vaccination the IgG and IgA titres against inner proteins of influenza A and B viruses tested with ELISA showed a distinct elevation in the vaccinated (HC and RA) vs. non-vaccinated groups. After vaccination, significantly increased median titres of HI for A (H1N1), A (H3N2) and B were observed in controls and in RA. Compared to adalimumab, etarnercept, tocilizumab, infliximab, methotrexate and leflunomide, rituximab-treated patients had the lowest % of patients with of HI elevation for all three antigens (44%, H1N1; 22% H3N2, 50% B), which confirmed previous seasonal flu vaccination data (1). Seroconversion (postvaccination antibody titers ≥40 in patients whose prevaccination titres were 180 days. Similarly, the seroresponse (seroconversion or fold increase in antibody titers ≥4 in patients whose prevaccination titers were ≥10) in RA patients did not persist >180 days. Conclusions The seroresponse observed in RA compared to controls was higher for A (H1N1) but not for A (H3N2) and B. We found that vaccination largely tilts the balance from negative ELISA IgG and IgA titres to positive titres in those RA patients who respond with H1N1 >4-fold titre elevation. The immune responses (for all antigens tested) of treated RA patients are higher than non-treated patients for all medications used, except rituximab. References Eisenberg R.A. et al. Rituximab-treated patients have a poorer response to influenza vaccination. J Clin Immunol. 2013 Feb; 33(2):388-396. Disclosure of Interest None declared