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The biomaterial-cells interface is one of the most fundamental issues in tissue regeneration. Despite many years of scientific work, there is no clear answer to what determines the desired adhesion of cells and the synthesis of ECM proteins. Crystallinity is a characteristic of the structure that influences the surface and bulk properties of semicrystalline polymers used in medicine. The crystallinity of polycaprolactone (PCL) was varied by changing the molecular weight of the polymer and the annealing procedure. Measurements of surface free energy showed differences related to substrate crystallinity. Additionally, the water contact angle was determined to characterise surface wettability which was crucial in the analysis of protein absorption. X-ray photoelectron spectroscopy was used to indicate oxygen bonds amount on the surface. Finally, the impact of the crystallinity, and related properties were demonstrated on dermal fibroblasts' response. Cellular proliferation and expression of selected genes: α-SMA, collagen I, TIMP, integrin were analysed.

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that is still not well understood in terms of its pathogenesis and presents diagnostic and therapeutic challenges. Monocytes are key players in initiating and maintaining inflammation through the production of pro-inflammatory cytokines and S100 proteins in RA. This study aimed to test a specific DNA methylation inhibitor (RG108) and activator (budesonide) in the regulation of pro-inflammatory mediators—especially the S100 proteins. We also searched for new biomarkers of high disease activity in RA patients. RNA sequencing analysis of healthy controls (HCs) and RA monocytes was performed. Genes such as the S100 family, TNF, and IL-8 were validated by qRT-PCR following DNA-methylation-targeted drug treatment in a monocytic THP-1 cell line. The concentrations of the S100A8, S100A11, and S100A12 proteins in the sera and synovial fluids of RA patients were tested and correlated with clinical parameters. We demonstrated that RA monocytes had significantly increased levels of S100A8, S100A9, S100A11, S100A12, MYD88, JAK3, and IQGAP1 and decreased levels of IL10RA and TGIF1 transcripts. In addition, stimulation of THP-1 cells with budesonide statistically reduced the expression of the S100 family, IL-8, and TNF genes. In contrast, THP-1 cells treated with RG108 had increased levels of the S100 family and TNF genes. We also revealed a significant upregulation of S100A8, S100A11, and S100A12 in RA patients, especially in early RA compared to HC sera. In addition, protein levels of S100A8, S100A11, and S100A12 in RA synovial fluids compared to HC sera were significantly increased. Overall, our data suggest that the S100A8 and S100A12 proteins are strongly elevated during ongoing inflammation, so they could be used as a better biomarker of disease activity than CRP. Interestingly, epigenetic drugs can regulate these S100 proteins, suggesting their potential use in targeting RA inflammation.

Magdalena Massalska,1 Marzena Ciechomska,1 Ewa Kuca-Warnawin,1 Tomasz Burakowski,1 Anna Kornatka,1 Anna Radzikowska,1 Dariusz Pawlak,2 Barbara Muz,3 Adrianna Loniewska-Lwowska,4 Andrzej Palucha,5 Pawel Maldyk,6,7 Wlodzimierz Maslinski1 1Department of Pathophysiology and Immunology, National Institute of Geriatrics, Rheumatology, and Rehabilitation (NIGRiR), Warsaw, 02-637, Poland; 2Department of Pharmacodynamics, Medical University of Bialystok, Bialystok, 15-222, Poland; 3Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO, 63108, USA; 4Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, 02-106, Poland; 5Genomed S.A. Ponczowa 12, Warsaw, 02-971, Poland; 6Department of Rheumoorthopaedic Surgery, National Institute of Geriatrics, Rheumatology, and Rehabilitation (NIGRiR), Warsaw, 02-637, Poland; 7Clinical Department of Orthopaedic and Traumatology of Locomotor System, Enfant-Jesus Clinical Hospital, Warsaw, 02-005, PolandCorrespondence: Magdalena Massalska, Department of Pathophysiology and Immunology, National Institute of Geriatrics, Rheumatology, and Rehabilitation, Spartanska 1, Warsaw, 02-637, Poland, Tel/Fax +48 22 670 94 94, Email magdalena.massalska@spartanska.plBackground: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery.Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls.Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls.Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.Keywords: rheumatoid arthritis, bone marrow, CTLA-4-Fc, indoleamine 2,3- dioxygenase, CD4+Foxp3+, monocytes

Dysregulation in type I IFN and IFN-stimulated genes (ISGs) induced by monocytes is one of the key features of systemic sclerosis (SSc) pathogenesis. Abnormalities in microRNA (miRNA) expression are related to excessive IFN production, however the role of miRNA remains largely elusive in SSc monocytes. This study explores global miRNA-mRNA profiling of SSc monocytes and functional attenuation of IFN and ISGs by specific miRNAs. Global sequencing of mRNA (mRNA-seq) and miRNA (miRNA-seq) samples were performed simultaneously on healthy controls and SSc monocytes. Following computational analysis, selected miRNAs-mRNA candidates were validated, correlated with clinical parameters, and tested by functional assays. Transcriptomics data and qPCR analysis confirmed IFN signature in SSc but not in rheumatoid arthritis monocytes. Based on miRNA-seq analysis, five miRNAs were selected for further validation. Only the expression patterns of miRNA-26a-2-3p and miRNA-485-3p were confirmed and negatively correlated with clinical parameters. Exogenous delivery of miRNA-26a-2-3p to TLR-stimulated monocytic THP-1 cells specifically inhibited ISGs but not inflammasome activity in functional assays. In conclusion, our miRNA-mRNA co-sequencing and functional analysis identify miRNA-26a-2-3p as a new candidate, which is predicated to negatively regulate ISGs. This implies that reduced expression of miRNA-26a-2-3 may be involved in pathogenic IFN signature in SSc monocytes.

Complex pathogenesis of systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is associated with an imbalance of various Th-cell subpopulations. Mesenchymal stem cells (MSCs) have the ability to restore this balance. However, bone marrow-derived MSCs of SLE and SSc patients exhibit many abnormalities, whereas the properties of adipose derived mesenchymal stem cells (ASCS) are much less known. Therefore, we examined the effect of ASCs obtained from SLE (SLE/ASCs) and SSc (SSc/ASCs) patients on Th subset differentiation, using cells from healthy donors (HD/ASCs) as controls. ASCs were co-cultured with activated CD4+ T cells or peripheral blood mononuclear cells. Expression of transcription factors defining Th1, Th2, Th17, and regulatory T cell (Tregs) subsets, i.e., T-bet, GATA3, RORc, and FoxP3, were analysed by quantitative RT-PCR, the concentrations of subset-specific cytokines were measured by ELISA, and Tregs formation by flow cytometry. Compared with HD/ASCs, SLE/ASCs and especially SSc/ASCs triggered Th differentiation which was disturbed at the transcription levels of genes encoding Th1- and Tregs-related transcription factors. However, we failed to find functional consequences of this abnormality, because all tested ASCs similarly switched differentiation from Th1 to Th2 direction with accompanying IFNγ/IL-4 ratio decrease, up-regulated Th17 formation and IL-17 secretion, and up-regulated classical Tregs generation.

Monocytes/macrophages play a central role in chronic inflammatory disorders, including rheumatoid arthritis (RA). Activation of these cells results in the production of various mediators responsible for inflammation and RA pathogenesis. On the other hand, the depletion of macrophages using specific antibodies or chemical agents can prevent their synovial tissue infiltration and subsequently attenuates inflammation. Their plasticity is a major feature that helps the switch from a pro-inflammatory phenotype (M1) to an anti-inflammatory state (M2). Therefore, understanding the precise strategy targeting pro-inflammatory monocytes/macrophages should be a powerful way of inhibiting chronic inflammation and bone erosion. In this review, we demonstrate potential consequences of different epigenetic regulations on inflammatory cytokines production by monocytes. In addition, we present unique profiles of monocytes/macrophages contributing to identification of new biomarkers of disease activity or predicting treatment response in RA. We also outline novel approaches of tuning monocytes/macrophages by biologic drugs, small molecules or by other therapeutic modalities to reduce arthritis. Finally, the importance of cellular heterogeneity of monocytes/macrophages is highlighted by single-cell technologies, which leads to the design of cell-specific therapeutic protocols for personalized medicine in RA in the future.

Rheumatoid arthritis (RA) affects around 1.2% of the adult population. RA is one of the main reasons for work disability and premature retirement, thus substantially increasing social and economic burden. Biological disease-modifying antirheumatic drugs (bDMARDs) were shown to be an effective therapy especially in those rheumatoid arthritis (RA) patients, who did not adequately respond to conventional synthetic DMARD therapy. However, despite the proven efficacy, the high cost of the therapy resulted in limitation of the widespread use and unequal access to the care. The introduction of biosimilars, which are much cheaper relative to original drugs, may facilitate the achievement of the therapy by a much broader spectrum of patients. In this review we present the properties of original biologic agents based on cytokine-targeted (blockers of TNF, IL-6, IL-1, GM-CSF) and cell-targeted therapies (aimed to inhibit T cells and B cells properties) as well as biosimilars used in rheumatology. We also analyze the latest update of bDMARDs’ possible influence on DNA methylation, miRNA expression and histone modification in RA patients, what might be the important factors toward precise and personalized RA treatment. In addition, during the COVID-19 outbreak, we discuss the usage of biologicals in context of effective and safe COVID-19 treatment. Therefore, early diagnosing along with therapeutic intervention based on personalized drugs targeting disease-specific genes is still needed to relieve symptoms and to improve the quality of life of RA patients.

Objective To explore global miRNA and transcriptomic profiling of monocytes from RA patients compared with healthy controls in order to predict which aberrantly expressed miRNA can negatively modulate inflammatory molecules. Methods Using next-generation sequencing, we have performed simultaneous global analysis of miRNA (miRNA-seq) and transcriptome (RNA-seq) of monocytes from RA patients and healthy controls. Global analysis of miRNA of SSc monocytes was also performed. Following differential analysis and negative correlation, miRNA–RNA pairs were selected. Results We found that 20 specific miRNA candidates are predicted to silence inflammatory mediators, out of 191 significantly changed miRNAs in RA monocytes. Based on the highest scoring in terms of negative correlation (r = −0.97, P = 1.75e–07, false discovery rate = 0.04) and the number of seeds in miRNA responsible for negative regulation, we selected miRNA-146b and its target gene anti-inflammatory retinoic acid receptor alpha (RARA). Similarly to next-generation sequencing, qPCR analysis also confirmed negative correlation between miRNA-146b and RARA expression (r = −0.45, P = 0.04). Additionally, miRNA-146b expression in RA monocytes significantly correlated with clinical parameters including DAS28 for RA with CRP (DAS28-CRP) and ESR (DAS28-ESR), whereas overexpression of miRNA-146b was able to functionally reduce RARA expression in the human monocytic cell line THP-1. Finally, circulating miRNA-146b expression in sera and SFs was significantly elevated in RA patients. Conclusions Overall, in this study we have identified a new miRNA-146b candidate that is predicted to negatively regulate the anti-inflammatory RARA transcript, whereas circulating miRNA-146b level can be used as a biomarker predicting pro-inflammatory RA progression and disease activity.

The development of biological disease-modifying antirheumatic drugs (bDMARDs) and target synthetic DMARDs (tsDMARDs), also known as small molecule inhibitors, represent a breakthrough in rheumatoid arthritis (RA) treatment. The tsDMARDs are a large family of small molecules targeting mostly the several types of kinases, which are essential in downstream signaling of pro-inflammatory molecules. This review highlights current challenges associated with the treatment of RA using small molecule inhibitors targeting intracellular JAKs/MAPKs/NF-κB/SYK-BTK signaling pathways. Indeed, we have provided the latest update on development of small molecule inhibitors, their clinical efficacy and safety as a strategy for RA treatment. On the other hand, we have highlighted the risk and adverse effects of tsDMARDs administration including, among others, infections and thromboembolism. Therefore, performance of blood tests or viral infection screening should be recommended before the tsDMARDs administration. Interestingly, recent events of SARS-CoV-2 outbreak have demonstrated the potential use of small molecule inhibitors not only in RA treatment, but also in fighting COVID-19 via blocking the viral entry, preventing of hyperimmune activation and reducing cytokine storm. Thus, small molecule inhibitors, targeting wide range of pro-inflammatory singling pathways, may find wider implications not only for the management of RA but also in the controlling of COVID-19.

In this study, we analysed the expression level of sera circulating miRNA-5196 in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients before and after tumor necrosis factor (TNF)-α therapy as biomarkers predicting positive treatment outcome. We enrolled 10 RA patients, 13 AS patients, and 12 healthy individuals in the study. The expression of miRNA-5196 was measured by real-time polymerase chain reaction before and after anti-TNF-α therapy. Disease activity of RA patients was assessed using disease activity score 28 (DAS28), whereas ankylosing spondylitis DAS (ASDAS) was used in AS patients. MiRNA-5196 expression was significantly higher in patients with RA and AS before TNF-α therapy than in those following anti-TNF-α therapy and healthy controls. Changes in miRNA-5196 expression positively correlated with delta DAS28 or delta ASDAS, respectively, following TNF-α therapy. In contrast, changes in C-reactive protein (CRP) levels in RA and AS patients did not positively correlate with DAS28 or ASDAS changes. Receiver-operating characteristic analysis showed better diagnostic accuracy of miRNA-5196 expression both in RA (area under curve (AUC) = 0.87, p = 0.055) and AS patients (AUC = 0.90, p = 0.050) compared to CRP levels in RA (AUC = 0.75, p = 0.201) and AS patients (AUC = 0.85, p = 0.086) upon biologic therapy treatment. Finding novel biomarkers, including miRNA-5196 which allow to predict and monitor anti-TNF-α response, would be of clinical value especially during the early phase of RA or AS development.

Rheumatoid arthritis (RA) is a long-term autoimmune disease of unknown etiology that leads to progressive joint destruction and ultimately to disability. RA affects as much as 1% of the population worldwide. To date, RA is not a curable disease, and the mechanisms responsible for RA development have not yet been well understood. The development of more effective treatments and improvements in the early diagnosis of RA is direly needed to increase patients’ functional capacity and their quality of life. As opposed to genetic mutation, epigenetic changes, such as DNA methylation, are reversible, making them good therapeutic candidates, modulating the immune response or aggressive synovial fibroblasts (FLS—fibroblast-like synoviocytes) activity when it is necessary. It has been suggested that DNA methylation might contribute to RA development, however, with insufficient and conflicting results. Besides, recent studies have shown that circulating cell-free methylated DNA (ccfDNA) in blood offers a very convenient, non-invasive, and repeatable “liquid biopsy”, thus providing a reliable template for assessing molecular markers of various diseases, including RA. Thus, epigenetic therapies controlling autoimmunity and systemic inflammation may find wider implications for the diagnosis and management of RA. In this review, we highlight current challenges associated with the treatment of RA and other autoimmune diseases and discuss how targeting DNA methylation may improve diagnostic, prognostic, and therapeutic approaches.

Background:Patients with rheumatic diseases such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) are at increased risk of developing dyslipidaemia and premature cardiovascular disease (CVD)(1).Objectives:To investigate the relationship between proinflammatory cytokines, microRNA, and lipid profile in patients with RA, AS, and PsA.Methods:A group of 65 patients (RA15/ AS25/ PsA25) with high disease activity (mean DAS28 5,98 / ASDAS-CRP 3,7/ DAPSA 38,5) and 25 healthy controls (HC) were compared. Lipid profile comprised triglycerides (TG), total cholesterol(TC), low (LDL), and high-density lipoprotein (HDL). Serum concentrations of IL-6, IL-21, IL-17, TNF and osteoprotegerine (OPG) were measured by commercially available enzyme-linked immunosorbent assays. Expression of miR-233-5p,miR-92-3p,miR-485-3p,miR-10b-5p,let-7d-5p, miR-26 -a-2-3p levels in sera was normalized to miR-16a internal control. The Mann-Whitney test was applied for intergroup comparison, the correlation was assessed using Spearman’s Rank test.Results:Patients with RA revealed mixed dyslipidemia (mean values:TC196; LDL 117; HDL, 48 TG; 124 mg/dl), PsA revealed hypertriglyceridemia (TC 175; LDL 100; HDL 50; TG137 mg/dl) and in AS no specific profile was found (TC 179; LDL98; HDL55; TG103 mg/dl). Higher expression of miR-485was observed in patients with PsA (4,8-fold) and AS (5,9-fold) compared to RA and HC groups (3,1 and 1,5-fold p=0,02). Similarly, miR-26a revealed higher expression in patients with PsA (28,4-fold) and AS (21,5-fold) than in RA and HC (3,5 and 2,9-fold pConclusion:Differences in proinflammatory profile in RA, PsA, and AS seem to be associated with different phenotypes of dyslipidemia. In PsA expression of miR-92b and higher levels of TNF, IL-17 and OPG are associated with altered lipid profile and hypertriglyceridemia. High activity of AS is associated with lowered expression of let-7d, possibly influencing TC and HDL levels. Therefore, measuring lipid profile in active disease might be misleading. In active RA increase of TC and LDL levels was associated with high IL-21 concentration, while hypertriglyceridemia with miR-92b and miR-26a expressions.References:[1]Bonek K,et al AB0775 Peripheral joint inflammation is associated with more proatherogenic cardiovascular risk profile in patients with psoriatic arthritis.Annals of the Rheumatic Diseases 2020;79:1685-1686Disclosure of Interests:None declared.

MicroRNA-146a (miR-146a) has been shown to play an important role in the regulation of inflammatory innate immune responses, and found to be differentially expressed in rheumatoid arthritis (RA). Through NF-κB pathway, this molecule is able to stimulate the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-17. It has been also suggested that single-nucleotide polymorphisms (SNPs) in miRNA sequences may alter miRNA expression and that miR-146a rs2910164 SNP may contribute to RA development. These observations prompted us to analyze the potential associations between the miR-146a-3p (rs2910164, G > C) and NFkB1 (rs28362491, ins/del ATTG) polymorphisms and miR-146a-5p expression in patients’ sera in relation to clinical outcome of the treatment as well as predisposition to RA. Genotyping was performed in 111 patients and 130 healthy individuals while 16 controls and 13 RA patients (before and after three months of therapy with TNF-α inhibitors (TNFi)) were studied for the circulating miR-146a-5p serum expression level. Patients carrying the NFkB1 ins/ins genotype were characterized by worse response to TNFi treatment (p = 0.023). In patients, before TNFi therapy, expression levels of miR-146a-5p were less (0.422 ± 0.171) as compared to those detected after three months of treatment (1.809 ± 0.658, p = 0.033) and observed for healthy controls (5.302 ± 2.112, p = 0.048). Moreover, patients with higher circulating miR-146a-5p levels after three months of TNFi administration were more frequently carrying the rs2910164-C allele (p = 0.032). These results support the hypothesis that miR-146a might be involved in pathogenesis of RA and imply that miR-146a-3p polymorphism may be associated with miR-146a-5p levels in serum after anti-TNF-α treatment.

Objective To investigate whether epigenetic changes can modulate monocytes to produce tissue inhibitor of metalloproteinases 1 (TIMP-1) via Fra-2 (an activator protein 1 [AP-1] family member), a novel downstream mediator that promotes fibrogenesis. Methods AP-1 transcription factors and TIMP-1 expression were measured in monocytes from systemic sclerosis (SSc) patients and healthy controls. Involvement of Fra-2 in the regulation of TIMP-1 following treatment with Toll-like receptor 8 (TLR-8) agonist was investigated using a luciferase activity assay and chromatin immunoprecipitation (ChIP) analysis. Expression of TIMP-1 and Fra-2 was determined in response to TLR-8 treatment and to different histone modifications, including 3′-deazaneplanocin (DZNep) and apicidin. Fibroblasts from healthy controls were cocultured with DZNep plus TLR-8-treated healthy control monocytes. Results Up-regulation of Fra-2 was detected in bleomycin-challenged mice and in skin biopsy samples from SSc patients. Enhanced expression of Fra-2 and TIMP-1 was correlated in SSc monocytes (P = 0.021). The expression of Fra-1 was significantly reduced (P = 0.037) in SSc monocytes. Inhibiting AP-1 activity reduced TIMP-1 production in TLR-8-stimulated monocytes from healthy controls and SSc patients. ChIP experiments revealed binding of Fra-2 to the TIMP-1 promoter. Stimulation with DZNep plus TLR-8 enhanced Fra-2 and TIMP-1 expression in healthy control monocytes, whereas TLR-8 plus apicidin repressed Fra-2 and TIMP-1 expression. Finally, healthy control monocytes treated with DZNep plus TLR-8 induced strong production of α-smooth muscle actin in dermal fibroblasts, which was inhibited by TIMP-1-blocking antibody. Conclusion These data demonstrate a novel role of histone demethylation induced by DZNep on Fra-2-mediated TIMP-1 production by monocytes in the presence of TLR-8 agonist. This consequently orchestrates the transdifferentiation of fibroblasts, a key event in the pathogenesis of SSc.

Background Monocytes are central to the initiation of inflammation and bone erosion in rheumatoid arthritis (RA). Therefore, understanding of molecular pathways regulation in monocytes, especially at the early stages of RA development, is of crucial importance as it may help to predict the progression to the full-blown disease. However, mechanisms of gene regulation in monocytes, particularly the pattern of microRNA molecules (miRNAs) expression which can be involved in regulation of several cellular inflammatory pathways, in early phases of RA (i.e. at undifferentiated arthritis-UA stage) are not fully characterized. Objectives To investigate the pattern of miRNAs expression in monocytes that could serve as new biomarkers for RA development. Methods Magnetically sorted monocytes from peripheral blood of 19 patients with UA served for total RNA isolation. Total RNA from both sample and reference was labelled with Hy3 and Hy5 fluorescent label, respectively, using the miRCURY LNA microRNA Hi-Power Labeling Kit (Exiqon, Denmark). Fluorescent labelled samples and a reference RNA sample were hybridized to the miRCURY LNA microRNA Array 7th Gen (Exiqon), followed by scanning by Microarray Scanner System (Agilent Technologies, USA). The image analysis was carried out using ImaGene 9 Software (Exiqon). Results Out of 19 patients with UA enrolled in the study, 12 were verified for diagnosis after 4 years of follow-up (7 patients did not respond to the call for re-evaluation). Four patients developed full-blown RA (UA→RA patients), 6 patients remained still in UA phase (UA→UA patients), 1 patient was diagnosed as having Sjogren9s Syndrome, and 1 undifferentiated connective tissue disease. Baseline characteristics of UA→RA vs UA→UA patients were as follows: age (median: 50.5, range 37–59 years vs 52.5, range 32–63 years), CRP (median: 9.0, range 1–22 mg/l vs 8.0, range 1–21 mg/l), ESR (median: 37.0, range 4–47 mm/h vs 34.0, range 15–44 mm/h) and swollen joins count (median: 3.0, range 1–9 vs 2.5, range 1–4). Following computational unsupervised analysis we identified 50 miRNAs in monocytes that have the largest variation across all patients samples. From these 50 miRNAs we selected several specific miRNA candidates on the basis of significantly changed expression in monocytes of UA→RA vs UA→UA patients. Predicted specific miRNAs targeting inflammatory genes in monocytes of UA→RA patients are: miR-483–3p (2.7-fold increased, p=0.009), miR-378d (4.1-fold decreased, p=0.0059), miR-371b-5p (52.8-fold increased, p=0.0381) miR-642b-5p (13.7-fold increased, p=0.0380), and miR-25–3p (1.8-fold decreased, p=0.0317). Additional validation of selected miRNAs candidates will be further performed using qPCR analysis. Conclusions Our results indicate new miRNA candidates differentially expressed in peripheral blood monocytes from patients with UA who subsequently developed RA, in comparison to patients with UA who did not progress to RA after 4 years follow-up. Acknowledgements Supported by NCN Poland (grant 2012/06/A/NZ5/00059). Disclosure of Interest None declared

Background Monocytes play an important role in autoimmune disease including rheumatoid arthritis (RA) or systemic sclerosis (SSc). Monocytes are the first immune cells which migrate from blood to the site of inflammation leading to tissue destruction due to enhanced proinflammatory cytokines secretion. It has been shown that abnormalities in microRNA (miRNA) expression are related to inflammatory cytokines production by T and B cells in several rheumatic diseases, however miRNAs have never been fully analysed in monocytes population. The aim of this study was to evaluate a global miRNAs profiling of monocytes from RA and SSc patients which are predicted to target proinflammatory genes. Materials and methods Total RNAs from CD14+ monocytes of 12 RA, 10 SSc patients and 10 HC were isolated. Thirty-two samples were barcoded using Small-RNA-Library-Set, pooled and sequenced on Illumina HiSeq 2000 platform. Expression of circulating miRNA-5196 was also measured in sera of 12 RA patients before and after 6 month TNF-α therapy and correlated (Spearman analysis) with disease activity score (DAS) 28. Results Based on next generation sequencing (NGS) we received 10 million reads from each sample. Following computational analysis we selected 14 specific miRNA candidates which are predicted to target inflammatory mediators out of 188 and 171 significantly changed miRNAs in monocytes of RA and SSc patients, respectively. Interestingly, miRNA-10–5p (targeting IRAK4) was 3.1-fold increased in SSc compared to RA (p Conclusions Our results identify new miRNA candidates which are predicated to regulate inflammatory genes in RA and SSc monocytes whereas miRNA-10–5p could be used as a biomarker exclusively characterising SSc. Also circulating miRNA-5196 can be used as a good predictor of anti-TNF-α treatment in RA patients. Supported by 2015/16/S/NZ6/00041 from National Science Centre, Poland and EMBO ST Fellowship.

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute-phase protein that is elevated up to 1000-fold in times of infection or inflammation. This acute-phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine-like manner, possibly through toll-like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin-6 (IL-6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL-6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA-induced IL-6 secretion and that this was also mediated through the TLR adaptor protein IL-1 receptor-associated kinase 4. The effect is nuclear factor-κB-mediated because blockade of nuclear factor-κB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over-expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL-6 signalling in systemic sclerosis via enhanced TLR2 signalling.

Systemic sclerosis is a connective tissue disease of unknown aetiology characterised by autoimmunity, inflammation, vascular abnormalities and ultimately fibrosis. Although great advances have been made in determining the molecular mechanisms of disease pathogenesis over the last decade, aided by new genetic screens, no current specific disease-modifying treatment is yet available. Epigenetics is defined as heritable changes that are not due to changes in DNA sequence, and there is at present intense research effort to understand the basic mechanisms of epigenetic regulation and how these impact diseases. Epigenetic modifications and dysregulation are associated now with autoimmune disease, inflammatory disease and cancer. In rheumatic diseases all three epigenetic processes are associated with various diseases including rheumatoid arthritis and systemic sclerosis. In systemic sclerosis much focus has been on microRNAs; however, other modifications including DNA methylation are emerging to have a key role. This review examines the role of epigenetics in systemic sclerosis and appraises the contribution of each modification and suggests that modulators of epigenetic changes may be a novel therapeutic option.

Fibrosis is a common and intractable condition associated with various pathologies. It is characterized by accumulation of an excessive amount of extracellular matrix molecules that primarily include collagen type I. IL-6 is a profibrotic cytokine that is elevated in the prototypic fibrotic autoimmune condition systemic sclerosis and is known to induce collagen I expression, but the mechanism(s) behind this induction are currently unknown. Using healthy dermal fibroblasts in vitro, we analyzed the signaling pathways that underscore the IL-6-mediated induction of collagen. We show that IL-6 trans signaling is important and that the effect is dependent on STAT3; however, the effect is indirect and mediated through enhanced TGF-β signaling and the classic downstream cellular mediator Smad3. This is due to induction of the bone morphogenetic protein (BMP) antagonist Gremlin-1, and we show that Gremlin-1 is profibrotic and is mediated through canonical TGF-β signaling.

Background Systemic sclerosis (SSc) is a chronic autoimmune disease characterised by tissue fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play an important role in early stages of SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction. However, the exact factors that contribute to chronic inflammation and subsequently fibrosis progression are still unknown. Material and methods The expression pattern of IL-8, TIMP-1, AP-1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist stimulation was investigated. Exogenous microRNA-5196, which is predicted to bind 3′UTR of Fra-2 gene, was delivered in order to reverse profibrotic phenotype in monocytes. Expression of circulating microRNA-5196 was correlated with SSc parameters. Results DZNep+TLR8 agonist stimulation enhanced profibrotic TIMP-1, IL-8 and ROS generation in HC and SSc monocytes. As opposed by the decrease of miRNA-5196 and antioxidant SOD1 expression in SSc monocytes. Exogenous delivery of microRNA-5196 reduced Fra2 and TIMP-1 expression suggesting that it may be used as a potential modulator of fibrogenesis in SSc. Circulating microRNA-5196 was significantly increased in SSc and positively correlated with CRP level but not with Rodnan skin score or ESR. Conclusions These results suggest that microRNA-5196 can be used as a potential biomarker characterising SSc. Overall, this study may open new possibilities for the development of microRNA-5196-based diagnostics and therapy in early phases of SSc. This article is protected by copyright. All rights reserved.

Systemic sclerosis is an autoimmune connective tissue disease in which T cells play a prominent role. We and others have previously demonstrated a role for T cell-derived IL-13 in mediating the induction of collagen in dermal fibroblasts and that blockade with IL-13 antibodies attenuates this increase. In this study we want to probe the signalling that underpins IL-13 mediated matrix deposition. Isolated dermal fibroblasts were incubated with recombinant IL-13 and gene expression by qRT-PCR was performed for collagen1A1 and TGF-β1. Small interfering RNA (siRNA) was used to knock down STAT6 and a small molecule inhibitor was also used to block this pathway. MiR-135b was transfected into fibroblasts plus and minus IL-13 to see if this miR plays a role. miR-135b was measured in systemic sclerosis fibroblasts isolated from patients and also in serum. Results showed that IL-13 increased collagen expression and that this is independent from TGF-β1. This is dependent on STAT6 as targeting this blocked induction. MiR-135b reduces collagen induction in fibroblasts and scleroderma fibroblasts have lower constitutive levels of the miR. We further demonstrate that miR135b is repressed by methylation and may include MeCP2. In conclusion we show that STAT6 and miR-135b regulate IL-13-mediated collagen production by fibroblasts.

The induction of an effective immune response requires the activation of CD4+ T lymphocytes by APCs. While DCs have been shown to be pivotal in this process, it is now apparent that optimal CD4+ T-cell activation also requires B-lymphocyte APC function. Along with the acquisition of soluble antigens, it is known that B cells also acquire membrane-tethered antigens. Recent reports have described the relocation of intracellular antigens to the cell surface following immunogenic apoptosis. This study was designed to determine whether B cells can acquire and present such antigens to CD4+ T cells. By targeting the model antigen tetanus toxin C fragment to various cellular locations, we show that antigen-specific B cells acquire intracellular antigens that have relocated to the surface of cells undergoing immunogenic apoptosis. Crucially, we also demonstrate that antigen-specific B cells acquiring relocated antigen from apoptotic targets are capable of efficiently inducing CD4+ T-cell activation. We propose that the acquisition and presentation of intracellular antigens that have relocated to the cell surface during immunogenic apoptosis represents a novel means by which antigen-specific B cells contribute to the generation of immunity.

To investigate whether epigenetic changes can modulate monocytes to produce tissue inhibitor of metalloproteinases 1 (TIMP-1) via Fra-2 (an activator protein 1 [AP-1] family member), a novel downstream mediator that promotes fibrogenesis.AP-1 transcription factors and TIMP-1 expression were measured in monocytes from systemic sclerosis (SSc) patients and healthy controls. Involvement of Fra-2 in the regulation of TIMP-1 following treatment with Toll-like receptor 8 (TLR-8) agonist was investigated using a luciferase activity assay and chromatin immunoprecipitation (ChIP) analysis. Expression of TIMP-1 and Fra-2 was determined in response to TLR-8 treatment and to different histone modifications, including 3'-deazaneplanocin (DZNep) and apicidin. Fibroblasts from healthy controls were cocultured with DZNep plus TLR-8-treated healthy control monocytes.Up-regulation of Fra-2 was detected in bleomycin-challenged mice and in skin biopsy samples from SSc patients. Enhanced expression of Fra-2 and TIMP-1 was correlated in SSc monocytes (P = 0.021). The expression of Fra-1 was significantly reduced (P = 0.037) in SSc monocytes. Inhibiting AP-1 activity reduced TIMP-1 production in TLR-8-stimulated monocytes from healthy controls and SSc patients. ChIP experiments revealed binding of Fra-2 to the TIMP-1 promoter. Stimulation with DZNep plus TLR-8 enhanced Fra-2 and TIMP-1 expression in healthy control monocytes, whereas TLR-8 plus apicidin repressed Fra-2 and TIMP-1 expression. Finally, healthy control monocytes treated with DZNep plus TLR-8 induced strong production of α-smooth muscle actin in dermal fibroblasts, which was inhibited by TIMP-1-blocking antibody.These data demonstrate a novel role of histone demethylation induced by DZNep on Fra-2-mediated TIMP-1 production by monocytes in the presence of TLR-8 agonist. This consequently orchestrates the transdifferentiation of fibroblasts, a key event in the pathogenesis of SSc.

Systemic sclerosis is an autoimmune disease characterised by vascular dysfunction, impaired angiogenesis, inflammation and fibrosis. There is no currently accepted disease-modifying treatment with only autologous stem cell transplant showing clinically meaningful benefit. The lack of treatment options reflects our lack of understanding of the precise molecular mechanisms occurring in the disease. Recent investigations have begun to decipher the molecular pathways underpinning the different aspects of the disease and may provide a rational clinical target(s). Uncovering the molecular mechanisms of the disease is important in understanding systemic sclerosis treatment. The aim of this review was to examine the current thinking in SSc pathogenesis and will offer novel areas for research which may yield novel therapeutics.

Accumulative evidence demonstrates the crucial role of evolutionary conserved Toll-like receptors (TLRs) in identifying microbial or viral compounds. TLRs are also able to recognise endogenous molecules which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases including systemic sclerosis (SSc). A classic feature of SSc, is vascular injury manifested as Raynaud's phenomenon and ischaemia of the skin, resulting in the release of endogenous TLR ligands during inflammation and local tissue damage. These locally released TLR ligands bind TLRs possibly complexed to autoantibodies, and initiate intracellular signalling pathways and may be one of the mechanisms that initiate and drive autoimmunity and subsequent fibrosis. Activation of the immune system results in interferon (IFN) sensitive gene transcription. There is also an IFN gene signature in SSc peripheral blood. TLRs may represent the link between immune activation, common in SSc, and tissue fibrosis. Therefore, a better understanding of the mechanisms of TLR-mediated pathogenesis and therapies targeting individual TLRs, may provide a more specific approach of treating multi-systemic autoimmune diseases. This review aims to integrate the current knowledge of TLR function in the autoimmune disorders with particular emphasis on SSc. We suggest the TLR system as a new therapeutic target.

Interleukin-6 is a classic pro-inflammatory cytokine needed to mount an effective immune response. It is secreted by a wide array of cell types, however, its target cells are more restricted, due to the fact that very few cells, except lymphocytes and hepatocytes, express the functional membrane IL-6 receptor. This therefore limits the amount of cells that can respond to IL-6. Transsignalling, the shedding of the membrane bound form of the IL-6 receptor (sIL-6R) into the local microenvironment, greatly increases the range of cells that can respond e.g. as part of a wound healing response necessary to restore the homeostatic balance. Therefore, tight regulation of IL-6 signalling must occur to stop an inappropriate wound healing response occurring. This review focusses on the role of IL-6 in inflammation and fibrosing conditions, with a particular emphasis on systemic sclerosis (SSc), a chronic autoimmune disease in which a classical hallmark of fibrosis occurs. This fibrosis, in particular the skin and internal organs, leads to contractures and internal organ failure respectively with potential fatal consequences. In this review we will discuss the biology of IL-6 in the context of fibrosing conditions such as SSc and argue why molecular targeting of IL-6 is a promising therapeutic target.

The induction of an effective immune response requires the activation of CD4+ T lymphocytes by APCs. While DCs have been shown to be pivotal in this process, it is now apparent that optimal CD4+ T-cell activation also requires B-lymphocyte APC function. Along with the acquisition of soluble antigens, it is known that B cells also acquire membrane-tethered antigens. Recent reports have described the relocation of intracellular antigens to the cell surface following immunogenic apoptosis. This study was designed to determine whether B cells can acquire and present such antigens to CD4+ T cells. By targeting the model antigen tetanus toxin C fragment to various cellular locations, we show that antigen-specific B cells acquire intracellular antigens that have relocated to the surface of cells undergoing immunogenic apoptosis. Crucially, we also demonstrate that antigen-specific B cells acquiring relocated antigen from apoptotic targets are capable of efficiently inducing CD4+ T-cell activation. We propose that the acquisition and presentation of intracellular antigens that have relocated to the cell surface during immunogenic apoptosis represents a novel means by which antigen-specific B cells contribute to the generation of immunity.

Background and objectives Systemic sclerosis (SSc) is a chronic multisystem autoimmune disease characterised by skin and internal organs fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play a role in SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction, which contribute to fibrosis progression and chronic inflammation. The exact factors driving TIMP-1, IL-8 and ROS secretion are still unknown. The aim of this study was to investigate the expression pattern of profibrotic IL-8, TIMP-1, AP1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist (ssRNA) stimulation. Materials and methods The expression of Fra2, IL-8 and TIMP-1 and anti-oxidant superoxide dismutase 1 (SOD1) was measured by qRT-PCR in stimulated and unstimulated HC (n = 14) and SSc (n = 17) monocytes. Generation of ROS was determined using luciferase based assay. Fra2 DNA-binding activity was measured in AP-1 transcription assay in monocytic U937 cell line following epigenetic and TLR8 modifications. The level of anti-fibrotic miRNA-5196, which is predicted to bind and inhibit 3’UTR of Fra-2 gene, was also determined in HC and SSc monocytes. Results Combination of DZNep+TLR8 enhanced Fra2 (2-fold, p = 0.02), TIMP-1 (2-fold) and IL-8 (7.87-fold, p Conclusions These data suggest that DZNep and TLR8 agonist are able to enhance pro-fibrotic TIMP-1, IL-8 and oxidative stress generation. As opposed by the decrease of anti-fibrotic miRNA-5196 and anti-oxidant SOD1 expression in SSc monocytes, which might be used as a potential modulators of fibrogenesis in SSc. Supported by Homing Plus grant/2013–8/4 from Foundation for Polish Science and UMO-2015/16/S/NZ6/00041 from National Science Centre, Poland.

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.

Background and objectives To investigate whether epigenetic changes induced by 3’deazaneplanocin - DZNep and TLR signalling pathway can modulate monocytes to produce tissue-inhibitor of metalloproteinase-1 (TIMP-1) via Fra2 (Fos-related antigen 2) activity, a novel downstream mediator promoting fibrogenesis. Methods AP-1 and TIMP-1 expression following TLR8 treatment was measured by qRT-PCR in monocytes from Systemic sclerosis (SSc) patients (n = 13) and healthy controls (HC) (n = 13). TIMP-1 promoter activity was measured in U397 monocytic cell line using luciferase reporter assay. The effect of DZNep treatment on inhibition of tri-methylation of lysine 27 on histone 3 was analysed by Western Blot. Expression of TIMP-1 and Fra2 was determined in response to DZNep. The functional effect of TLR8 and DZNep-treated HC monocytes was studied on dermal fibroblasts’ trans-differentiation. Results Increased Fra2 and TIMP-1 expression was correlated in SSc monocytes (p = 0.021), but not in HC monocytes upon TLR8 stimulation. In contrast, the expression of anti-fibrotic Fra1 was significantly (p = 0.037) reduced in SSc monocytes compared to HC. Inhibiting AP-1 activity reduced TIMP-1 production in TLR8 stimulated HC and SSc monocytes. Also, TLR8 stimulation induced significant (p = 0.015) TIMP-1 promoter activity in monocytic U937 cells. Combination of DZNep plus TLR8 enhanced Fra2 (4.1 times) and TIMP-1 (4.7 times) expression in HC monocytes. However, the reverse effect on Fra2 and TIMP-1 expression was observed in SSc monocytes following stimulation. Finally, DZNep plus TLR8-treated HC monocytes induced strong production of collagen and a-SMA in dermal fibroblasts reflecting their trans-differentiation, which is a key event in the pathogenesis of SSc. Conclusions These data demonstrate that histone modification induces by DZNep has an opposite effect of Fra2-mediated TIMP-1 production on HC versus SSc monocytes. Therefore, DZNep cloud be used as a selective regulator of downstream mediators, which orchestrates SSc development.

Background and Objectives Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by fibrosis of skin and internal organs via accumulation of extra cellular matrix (ECM) proteins. This ECM deposition is observed when the homeostasis between matrix metalloproteinases (MMPs) and their natural inhibitors, eg. tissue inhibitor of metalloproteinases (TIMPs), is lost. MicroRNAs (miRs) are small regulatory RNAs that control gene expression by targeting 3’untranslated region (3’UTR) of various genes resulted in gene suppression. There is increasing evidence that miRs are key regulators of genes involved in fibrosis development. In particular, miR-29a was demonstrated to regulate ECM-associated genes in SSc patients. However, a better understanding of how epigenetic modulation by miR-29a prevents pathogenesis during SSc development is not fully defined. Materials and Methods Healthy control (HC) or SSc fibroblasts were cultured from skin biopsies. In some experiments, fibroblasts were transfected with miR-29a or control non-targeting miRNA and collagen, TIMP-1, MMP-1 and TGF-b activated kinase 1 binding protein 1 (TAB1) expression were measured using ELISA, qRT-PCR, Western Blotting, or functional collagen gel assay. In addition, HeLa cells (easy-to-transfect cells) were transfected with 3’UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors. Target protectors are designed to specifically interfere with TAB1 seed region, therefore are able to block miR-29a activity. Results In this study, we found that TAB1 is a novel target gene of miR-29a that subsequently leads to decreased TIMP-1 expression. In particular, using computational approach and 3’UTR of TAB1 construct, we demonstrated that TAB1 is a bona fide target of miR29a. Furthermore, we showed that miR-29a not only leads to TIMP-1 downregulation, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition (Oxozeanol) or TAB1 knockdown (siRNA against TAB1) resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. In addition, using Target protectors, TAB1 and TIMP-1 activity were derepressed due to miR-29a inhibition. Finally, miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via down regulation of TIMP-1 and collagen expression. Conclusions This study demonstrates that miR-29a directly represses its novel target gene - TAB1 in dermal fibroblasts, which results in decreased TIMP-1. Exogenous miR-29a was also able to reverse profibrotic phenotype of SSc fibroblasts, thus highlighting the potential therapeutic role of miR-29a in SSc and fibrogenesis in general.

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells. We and others have shown that monocytes along with fibroblasts play an important role in the production of pro-fibrotic factors such as IL-6 and TIMP-1 (tissue-inhibitor of metalloproteinase-1). Importantly, the balance between TIMP-1 and MMPs is altered in SSc leading to abnormal ECM formation. However, the exact factors which drive pro-fibrotic TIMP-1 in monocytes have not been fully established. Previous studies showed that AP-1 transcription factors including Fra-2 and JunD are involved in TIMP-1 gene expression in hepatic stellate cells contributing to liver fibrosis. Also Fra-2 transgenic mice develop a proliferative vasculopathy of the lung and skin fibrosis resembling similar disease manifestations seen in SSc patients. Objectives The aim of this study is to determine the role of Fra-2 in TIMP-1 production. We also plan to determine if TIMP-1 is a direct target gene of Fra-2 upon TLR8 stimulation in SSc monocytes. Additionally, we want to investigate if altered Fra-2 signalling results in enhanced TIMP-1 production thus contributing to ECM deposition and fibrosis development observed in systemic sclerosis. Methods 20 patients with SSc, one IRAK4 deficient patient and 20 HC (healthy control) were included in this study. Peripheral blood monocytes were further separated by CD14+ microbeads. Expression of TIMP-1 and Fra-2 by monocytes was determined by ELISA, qRT-PCR in response toTLR8 agonists (ssRNA). Results The results from our group demonstrated that circulating monocytes from SSc patients contribute to the imbalance between TIMP-1 and MMPs and to an increased pro-fibrotic IL-6 production upon TLR8 agonist stimulation (ssRNA). Interestingly, monocytes from an IRAK-4 (a key kinase in TLR-signalling) deficient patient did not produce IL-6 and TIMP-1. In addition, the results showed that the basal gene expression level of Fra-2 in SSc monocytes is higher than in healthy or IRAK-4 deficient monocytes. Interestingly, TLR8 activation induced even stronger Fra-2 gene expression and correlated with increased expression of TIMP-1 in SSc monocytes, suggesting that the TLR signalling pathway may be involved in Fra-2 mediated TIMP-1 induction and therefore promotes fibrosis. Also, we plan to pre-treat monocytes with the chemical AP-1 inhibitor (T-5224) and assess both gene and protein level of TIMP-1 upon TLR8 activation. Conclusions This study helps to understand the molecular mechanisms of Fra-2 mediated fibrogenesis in SSc. Disclosure of Interest None Declared

Background Systemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells including monocytes. It was suggested that monocytes along with fibroblasts play an important role in the production of profibrotic factors such as IL-6 and TIMP-1 (tissue-inhibitor of metalloproteinase-1). TIMPs are specific inhibitors of matrix metalloproteinases (MMPs) regulating extracellular matrix (ECM) turnover. Importantly, the balance between TIMPs and MMPs is altered in most pathological stages including SSc and is associated with abnormal ECM formation. However, the exact factors which drive both profibrotic TIMP-1 and IL-6 secretion have not been fully established. Objectives We aim to test whether circulating monocytes from SSc patients and dermal fibroblasts produce TIMP-1 and IL-6 in response to TLR activation, which contributes to excessive matrix deposition and consequently disease progression. Methods 23 patients with SSc and 19 HC (healthy control) were included in this study. Peripheral blood monocytes were further separated by CD14+ microbeads. Production of TIMP-1, IL-6 by monocytes and dermal fibroblasts was determined by ELISA, qRT-PCR or functional assay in response to either panel of conventional TLR agonists or Danger Associated Molecular Patterns (DAMPs) such as High-Mobility Group Box-1 protein (HMGB-1) and hyaluronic acid (HA). Results TIMP-1 produced by monocytes was elevated in SSc patients compared to HC as measured by qRT-PCR (fold increase 3.1, p=0.03) and ELISA (fold increase 3.2, p=0.02). The level of TIMP-1 present in SSc sera patients was also higher than in the HC (fold increase 1.8, p Conclusions This study indicates a potential link between TLR signaling and excessive TIMP-1 and IL-6 secretion in circulating monocytes from SSc patients and activated dermal fibroblasts, supporting an important role of these cells in production of pro-fibrotic factors. Disclosure of Interest None Declared

Interleukin-6 (IL-6) is a classic pro-inflammatory cytokine critical in mounting an effective immune response. It is secreted by a wide array of cell types; however, its effector cells are more restricted, owing to the fact that very few cells, except lymphocytes and hepatocytes, express the functional membrane IL-6 receptor thus reducing the number of IL-6-responsive cells. Trans-signalling, the shedding of the membrane-bound form of the IL-6 receptor into the local microenvironment, greatly increases the range of cells that can respond. IL-6 has been demonstrated to have a pivotal role in the pathogenesis of rheumatoid arthritis, Castleman's disease and Crohn's disease exemplified by the use of an anti-IL-6 biological therapy. However, IL-6 is also associated with the autoimmune disease systemic sclerosis (SSc) and has been shown to be directly fibrotic. Elevated levels of IL-6 are found in SSc patients and this correlates with skin thickness, suggesting a causal effect. This review focuses on the role of IL-6 in SSc, a chronic autoimmune disease with fibrosis. In particular, we will examine the evidence base of the role of IL-6 in fibrosis in this condition, especially the downstream effector pathways. We will then argue why molecular targeting of IL-6 is a promising therapeutic target in this fibrosing disease.

Background and Objectives Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterised by inflammation and fibrosis. The fibrosis is due to an accumulation of extracellular matrix material due to enhanced deposition and/or reduced degradation. We and others have shown that abnormal production of TIMP-1 may contribute to fibrosis in SSc. Acute Serum Amyloid A (A-SAA) is an acute phase reactant and its levels correlate with ESR and CRP thus it is relevant in inflammatory conditions and has been demonstrated to be elevated in serum from SSc patients. The aim of this study was to determine the effects of A-SAA on dermal fibroblasts IL-6 and TIMP-1 secretion and its signalling mechanism downstream of ligation. Materials and Methods Human healthy dermal fibroblasts from punch biopsies were cultured at low passage and then treated with recombinant A-SAA (10 ug/ml) for 24 hours. After the set time points the cells were harvested and the supernatant removed. ELISAs were performed in triplicate for IL-6 TIMP-1, and MMP-1 levels. In some experiments cells were pretreated with 100 uM of WRW4, a specific formyl peptide receptor inhibitor with A-SAA. Also we used fibroblasts derived from a patient with a genetic non sense mutation that results in no IRAK-4 protein production and hence a halt in TLR signalling. IRAK-4 is a central downstream mediator of Toll-Like Receptor mediated signalling and is crucial for such signalling. Results Healthy human dermal fibroblasts incubated with A-SAA secreted high levels of IL-6 compared to untreated control cultures. Moreover A-SAA induced increased levels of TIMP-1 both at the mRNA levels and also the protein levels as determined by ELISA. Levels of the target of TIMP-1, MMP-1 protein levels were not altered at all. Thus the effect leads to a shift in the ratio of TIMP-1 to MMP-1 favouring ECM deposition. Pretreatment with WRW4 prior to A-SAA did not alter IL-6 or TIMP-1 expression levels, showing that the formyl peptide receptor plays no role in TIMP-1 induction mediated by addition of A-SAA. Furthermore cells derived from a gene deleted IRAK-4 patient with no IRAK-4 protein stimulated with A-SAA compared to control fibroblasts had TIMP-1, IL-6 levels compared to non-treated (media alone) dermal fibroblasts. Conclusions A-SSA induces TIMP-1, but importantly does not alter levels of TIMP-1 target MMP-1, thus shifting the TIMP-1/MMP-1 ratio. IL-6, a classic proinflammatory cytokine involved in SSc pathogenesis, is also elevated by A-SAA treatment in vitro. The signalling involved IRAK-4, a critical downstream messenger of TLR mediated signalling, but not formyl peptide receptors.

Background and Objectives Systemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells including monocytes. Monocytes along with fibroblasts play an important role in the production of profibrotic factors such as IL-6 and TIMP-1 (tissue-inhibitor of metalloproteinase-1). TIMPs are specific inhibitors of matrix metalloproteinases (MMPs) regulating extracellular matrix (ECM) turnover. Importantly, the balance between TIMPs and MMPs is altered in most pathological stages including SSc and is associated with abnormal ECM formation. However, the exact factors which drive both profibrotic TIMP-1 secretion are not fully defined. We aim to test whether circulating monocytes from SSc patients produce TIMP-1 in response to TLR activation and serum factors, which contributes to excessive matrix deposition and consequently disease progression. Materials and Methods 25 patients with SSc, one IRAK4 deficient patient and 20 HC (healthy control) were included in this study. Peripheral blood monocytes were further separated by CD14+ microbeads. Production of TIMP-1, IL-6 by monocytes was determined by ELISA, qRT-PCR or functional assay in response to either panel of conventional TLR agonists or HC and SSc sera. Skin section from SSc patient was stained with CD14+ and TIMP-1 antibodies and further analysed by confocal microscopy. Results TIMP-1 production by monocytes was observed in the SSc skin section and was upregulated in SSc patients compared to HC. Incubation of HC monocytes with SSc sera resulted in functionally active TIMP-1 production. However, pre-treatment with MyD88 inhibitor, but not control peptide, decreased TIMP-1 secretion. Furthermore, SSc-mediated TIMP-1 induction by monocytes was attenuated when FcγR was blocked and also when SSc sera were treated with DNA/RNA endonuclease prior to stimulation. This indicates that SSc sera contain RNA/DNA agonists inducing TIMP-1 production. Indeed, direct treatment of HC and SSc monocytes with a panel of TLR ligands demonstrated strong TIMP-1 and IL-6 production following triggering with TLR8 agonists (ssRNA). TLR8-mediated TIMP-1 production was reduced in monocytes from a patient with a genetic TLR signalling defect or HC monocytes cultured with MyD88 inhibitory peptide. Furthermore, matrix assay of TLR8 stimulated monocytes also confirmed functional TIMP-1 secretion, as matrix metalloproteinase-1 activity was significantly inhibited. Conclusions This study indicates a potential link between SSc serum factors and TLR signalling resulting in excessive TIMP-1 secretion by circulating monocytes from SSc patients.