Search

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.

Systemic inflammatory rheumatic diseases are considered as autoimmune diseases, meaning that the balance between recognition of pathogens and avoidance of self-attack is impaired and the immune system attacks and destroys its own healthy tissue. Treatment with conventional Disease Modifying Antirheumatic Drugs (DMARDs) and/or Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) is often associated with various adverse reactions due to unspecific and toxic properties of those drugs. Although biologic drugs have largely improved the outcome in many patients, such drugs still pose significant problems and fail to provide a solution to all patients. Therefore, development of more effective treatments and improvements in early diagnosis of rheumatic diseases are badly needed in order to increase patient’s functioning and quality of life. The reversible nature of epigenetic mechanisms offers a new class of drugs that modulate the immune system and inflammation. In fact, epigenetic drugs are already in use in some types of cancer or cardiovascular diseases. Therefore, epigenetic-based therapeutics that control autoimmunity and chronic inflammatory process have broad implications for the pathogenesis, diagnosis, and management of rheumatic diseases. This review summarises the latest information about potential therapeutic application of epigenetic modification in targeting immune abnormalities and inflammation of rheumatic diseases.

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 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.

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.

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.

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.

Background and objectives Recent experiments propose an essential role for B cells in the development of several autoimmune diseases either as autoantibody-producing cells or an antigen presenting cells (APC). However, the exact contribution of B cells in the early stages of rheumatoid arthritis (RA) remains unknown. One candidate autoantigen in RA is aggrecan, which, together with the disaccharide polymer hyaluron (HA), forms a major structural component of cartilage as part of an extracellular matrix (ECM). As B cells have been shown to be capable of acquiring antigens tethered to the surface of other cells, it is also possible that ECM tethered components may also be potential targets for joint-specific B cells. Therefore, we aimed to test the hypothesis that autoreactive B cells are important in the initiation of RA due to their unique ability to acquire integral joint autoantigens such as aggrecan leading to the activation of autoreactive T cells and subsequently disease progression. Materials and methods To monitor the acquisition of aggrecan we generated aggrecan-specific B cells. We have then addressed whether antigen-specific B cells are able to extract aggrecan tethered via HA to a non-internalisable surface measured by flow cytometry and T cell activation assays. Results We show, as expected, that aggrecan-specific B cells are able to present soluble aggrecan to CD4 T cells approximately 10 3 –10 4 times more efficiently than non-specific B cells. Based on kinetic analysis, we also demonstrate that aggrecan-specific B cells are able to acquire HA-tethered aggrecan from an immobilised surface within periods as short as 1 h. Furthermore, we show that aggrecan acquisition by specific B cells leads to efficient autoreactive T cell activation. Finally we have also extended this novel assay to mimic the synovial ECM, where aggrecan-specific B cells can extract aggrecan directly from nasal cartilage explants. Conclusion We demonstrate that antigen-specific B cells can acquire immobilised aggrecan leading to the stimulation of autoreactive CD4 T cells. These findings support the hypothesis that B cells may play an essential APC role in the early stage of joint pathogenesis. Therefore, a better understanding of these early processes could lead to define novel targets for therapy suitable in the early phase of RA.