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Background:TNF-mediated fibroblast-like synoviocyte (FLS) activation is important for inflammation and joint destruction in rheumatoid arthritis (RA). The role of TNF-receptor 1 (TNFR1) in FLS activation has thoroughly been characterized. The functions of TNFR2 are, however, largely unknown.Objectives:To investigate the contribution of TNFR2 to the TNF-mediated activation of FLS.Methods:RA-FLS were transfected with TNFR2-targeting siRNA pools and transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to confirm the RNA-seq results and to gain insights into the pathways that regulate TNFR2-mediated changes in FLS.Results:TNF stimulation of FLS resulted in a strong upregulation of proinflammatory cytokines, chemokines, tissue-degrading enzymes and other genes that are associated with synovial inflammation in RA. Silencing of TNFR2 markedly diminished the TNF-response of RA-FLS. Especially, “interferon”-stimulated-genes (ISGs) including putative master regulators of joint inflammation, such as the CXCR3 chemokines CXCL9, CXCL10 and CXCL11 were affected by the knockdown of TNFR2. Consistently, immunoblots showed that TNFR2 was required for the TNF-induced phosphorylation of the transcription factor STAT1, which is known to mediate the transcription of ISGs, such as CXCR3 chemokines.Conclusion:TNFR2 regulates proinflammatory gene expression in RA-FLS via STAT1 and thereby contributes to the detrimental effects of TNF in synovial joint inflammation.Disclosure of Interests:None declared more...

Objectives: The forkhead family transcription factor Foxp3 currently represents the most specific marker molecule for CD4 + CD25 + T cells with suppressive/regulatory capacity (Treg) in the mouse. Recent studies in the human system, however, indicate that the expression of Foxp3 can be T cell activation dependent. This tempted us to evaluate the significance of Foxp3 expression under autoimmune conditions with chronic T cell activation in patients with systemic lupus erythematosus (SLE) as compared with healthy controls (HCs). Methods: Proportions of peripheral blood CD4 + Foxp3 + T cells and CD4 + CD25 high T cells were determined in patients with active and inactive SLE as compared with HC by flow cytometry. Comparative analysis of the percentage of CD4 + Foxp3 + T cells and of percentage of CD4 + CD25 high T cells with clinical disease activity and T cell activation marker molecule expression were performed. Finally, the induction of Foxp3 expression was analysed upon T cell activation in vitro. Results: Proportions of CD4 + Foxp3 + T cells were significantly increased in patients with SLE as compared with HC and a significant correlation was observed between clinical disease activity and proportions of CD4 + Foxp3 + T cells. On the other hand, proportions of CD4 + CD25 high were decreased in SLE and no correlation with a T cell activation marker expression of was observed. In addition, in vitro activation of T cells induced Foxp3 expression. Conclusions: Our data suggest that the expression of Foxp3 on CD4 + T cells in patients with SLE, at least to some extent, reflects the activation of CD4 + T cells due to underlying disease activity and does not necessarily indicate a functional regulatory T cell capacity. more...

Background and objectives The heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in post-transcriptional regulation of gene expression. It has been shown to be highly upregulated in synovial tissue of patients with rheumatoid arthritis (RA). In addition, autoantibodies and T cells directed against hnRNP-A2/B1 can be found in RA patients. Recently, it was shown that silencing of hnRNP-A2/B1 in two animal models of RA, namely Collagen-induced arthritis and K/BxN serum transfer arthritis, led to reduction of arthritis severity. 1 To further elucidate the role of hnRNP-A2/B1 in RA, we sought of analysing the signalling pathways affected by silencing of hnRNP-A2/B1 in human fibroblast-like synoviocytes (FLS). Materials and methods siRNA-mediated silencing of hnRNP-A2/B1 in FLS was achieved by lipofectamine-based transfection. After three days, successful reduction of hnRNP-A2/B1 expression was analysed by real-time quantitative polymerase chain reaction (RT-qPCR). The role of hnRNP-A2/B1 in FLS was investigated by activating cells with TNF-α. Proteome Profiler Arrays were used to analyse cytokine production and phosphorylation of various signal transduction molecules. Interleukin (IL) -6 and IL-8 secretion was assayed using enzyme-linked immunosorption assay (ELISA). Results Silencing of hnRNP-A2/B1 led to a reduction of phosphorylation of AKT and mammalian target of rapamycin (mTOR) in TNF-α stimulated cells and a slight reduction in phosphorylation of p70 S6 kinase, which is a downstream signalling component of mTOR. Moreover, down-regulation of hnRNP-A2/B1 led to reduced levels of phosphorylated MAPK14 (p38α), and a reduction of MSK2 phosphoryl ation. Analysis of supernatants revealed reduced levels of CCL5 (RANTES), CXCL10 (IP-10), CCL20 (MIP-3α) and Serpine E1, but increased levels of ICAM-1. In addition, reduced secretion of Dickkopf-1 (DKK-1) or IGFBP-3 could be detected in silenced cells. Surprisingly, secretion of IL-6 and IL-8 was slightly increased in hnRNP-A2/B1 silenced FLS. Conclusions hnRNP-A2/B1 seems to play an important role in regulation of several signalling pathways, mainly the mTOR pathway, which is involved in translation and cell growth. Further analyses will be needed to fully understand the role of hnRNP-A2/B1 in signalling pathways operative in FLS and other inflammatory cell types involved in the pathogenesis of RA. Reference Herman S, et al . Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis. Arthritis Rheumatol 2005;67:2536–46 more...

Background The synovial membrane of diarthrodial joints is primarily composed of fibroblast-like synoviocytes (FLS) that form a complex tissue via discrete cell-to-cell connexions with wide intercellular matrix spaces. The distinct synovial tissue function that is crucial to joint homeostasis depends upon integrated activity of individual FLS. Using a in-vitro synovial organ culture system, we explore mechanisms of FLS cellular cooperation with special interest in the cell-to-cell transfer of mitochondria via interconnecting membrane nanotubes. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were cultured in spherical matrigel micromasses with an average size of 2 mm O. Data was acquired by confocal live cell imaging. Analysis of the resulting 4D movies was done with Imaris® software. Results To examine whether or not FLS transfer cytoplasmic cargo, we labelled 50% of FLS with red cell tracker dye and loaded the other 50% with green non-degradable microspheres. In a time series (8 days), we found that microspheres appear in red labelled cells. First evidence was found on Day 1 and over the course of the following days microspheres accumulated in red labelled cells with a transfer rate of 10% of newly affected cells/day. With special interest in the transfer of mitochondria, we repeated similar experiments with labelled mitochondria. We found that the transfer rate for these organelles is similar to the one for microspheres. Additionally, we were able to capture evidence that FLS indeed use their nanotube connexions for transfer of mitochondria. Conclusions Our experiments suggest transfer of cytoplasmic cargo, including organelles such as mitochondria, between FLS. These studies may provide insight into how synoviocytes orchestrate their activity. Further studies will demonstrate the significance of directed cargo exchange for cellular cooperation and the function of the normal as well as the diseased synovium. more...

Background The synovium is primarily built by fibroblast-like synoviocytes (FLS) that are connected to one another via cellular processes, thus forming a continuous network of cells. Its multicellularity requires precise coordination between cells to bring forth specialised tissue functions critical to joint homeostasis. Cell-to-cell transfer of cytoplasmic cargo may provide a means for intercellular communication, thereby facilitating the concerted behaviour of FLS. Using a 3D in-vitro model of the synovium, we analysed FLS capacity for exchange of cytoplasmic content. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were cultured in spherical matrigel micromasses with an average size of 2 mm O. For confocal live cell imaging and transmission electron microscopy, FLS were loaded with fluorescent non-degradable microspheres and labelled with fluorescent membrane dyes. Analysis of the resulting 4D movies was done with Imaris® software. Results To examine intercellular cytoplasmatic transfer, we labelled 50% of FLS with red cell tracker dye and loaded the other 50% with green microspheres. In a time series (8 days), we found that microspheres do indeed appear in red labelled cells. First evidence was found on Day 1 and over the course of the following days microspheres accumulated in red labelled cells with a transfer rate of 10% of newly affected cells/day. A similar experiment in 2D demonstrated microsphere movement within interconnecting membrane nanotubes. Transfer rates for microspheres into red cells were identical. Transmission Electron Microscopy (TEM) identified extracellular vesicles (exosomes) in discrete regions of intimate cell-to-cell contact but also open interconnecting tubes, suggesting distinct modes for transfer. Conclusions These studies reveal transfer of cytoplasmic cargo between FLS and may provide insight into how the synovial tissue operates. Further studies will demonstrate the significance of directed cargo exchange for cellular cooperation and the function of the normal as well as the diseased synovium. more...

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue where they integrate into the synovial lining network of fibroblast-like synovial cells (FLS). Here we analyse the dependence of Mo – FLS interaction on proinflammatory cytokines and the effect of anti-IL-6 antibody (Ab) Tocilizumab using real time confocal/multiphoton microscopy of 3D micromass tissue cultures. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14 + Mo were isolated from peripheral blood by magnetic bead sorting. For confocal imaging fluorescent labelled FLS and Mo were cultured in spherical extracellular matrix micromasses. Micromass cultures were set up in the presence of Tocilizumab or control Ab and stimulated with TNF-alpha. The resulting 4D live cell imaging movies were analysed with Imaris ® Software. Results The highest migratory activity of Mo was observed on days 4–7 of culture, upon the formation of a FLS lining layer around a 3D micromass, The presence of Tocilizumab in TNF-alpha stimulated cultures i) increased the proportion of migrating Mo from 12% in control cultures to 22%, ii) increased the maximum track length of migrating monocytes and iii) stimulated a fast oscillating movement pattern of Mo. Conclusions The 3D synovial tissue culture system allows for monitoring and analyses of subtle migration patterns of Mo in relation to the organised synovial lining architecture that depends on the presence of proinflammatory cytokines. Tocilizumab causes significant changes in the migratory behaviour of Mo that might lead to the release of Mo from the inflamed synovial tissue and contribute to the dissolution of inflammation. Ongoing experiments will address the molecular mechanism (s) of Mo – FLS interaction as well as the cellular source and sequence of cytokine production in order to identify potential targets for future therapeutic interventions in arthritis. more...

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue of arthritic joints. Monocyte migration appears to be associated with the expanding synovial lining network of fibroblast-like synovial cells (FLS). Whether cognitive interaction between Mo and FLS is required for an orchestrated migratory behavior has not been analyzed so far. Objectives We analyzed Mo migratory activity under inflammatory conditions in regard to cell-cell interactions with FLS. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14 + Mo were isolated from peripheral blood by magnetic bead sorting. FLS and Mo were labeled with fluorescent membrane dyes and cultured in spherical extracellular matrix micromasses with an average size of 1.5 mm for up to two weeks. For stimulation experiments, micromasses were cultured in medium containing 10 ng/ml of tumor necrosis factor (TNF). At different time-points cell migration was monitored in individual micromasses by real-time confocal microscopy. Results Cell migration could be subdivided into three successive phases of cell movement. Phase I (day 1-3 of culture) was characterized by the formation of the synovial lining layer. Mo in close contact with FLS appeared sessile. On average 20% of Mo were in no apparent contact with FLS and displayed a mobile and seeking behavior. During phase II (day 3-7) already >95% of Mo were in contact with FLS. The majority of Mo remained sessile whereas a fraction of Mo displayed a directed cell movement with an impressive maximum speed of up to 15 mcm/min. In addition the formation of Mo cell clusters was observed. The rapid Mo migration finally ceased during phase III (day 7-14). The addition of TNF i) increased the frequency and size of Mo cell clusters during phase II two and ii) prolonged the mobility of Mo into phase III. Conclusions The 3D synovial tissue culture system allows to monitor and analyzed subtle migration patterns of Mo in relation to the organized synovial lining architecture. Ongoing experiments address molecular mechanism(s) of Mo – FLS interaction in order to identify potential targets for future therapeutic intervention in arthritis. Disclosure of Interest None Declared more...