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Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Neutrophilic granulocytes and monocytes (granulomonocytic cells; GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely characterized. Here we have analyzed GMC in the murine collagen-induced arthritis (CIA) model of RA using multi-photon real time in vivo microscopy together with ex vivo analysis of GMC in tissue sections. GMC were abundant as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue. In addition, we observed the frequent formation of cell clusters consisting of both neutrophilic granulocytes and monocytes that actively contributed to the inflammatory process of arthritis. Treatment of animals with a single dose of prednisolone reduced the mean velocity of cell migration and diminished the overall immigration of GMC. In summary, our study shows that the combined application of real time in vivo microscopy together with elaborate static post-mortem analysis of GMC enables the description of dynamic migratory characteristics of GMC together with their precise location in a complex anatomical environment. Moreover, this approach is sensitive enough to detect subtle therapeutic effects within a very short period of time.

Objective The binding of abatacept (CTLA-4Ig) to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T cell activation. In addition, a direct effect of CTLA-4Ig on antigen-presenting cells (APCs) could contribute to the therapeutic effect. To further elucidate the mechanism of CTLA-4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid arthritis (RA) before and after the initiation of CTLA-4Ig therapy. Methods Peripheral blood mononuclear cells were analyzed before and at 2 and 4 weeks after the initiation of CTLA-4Ig therapy. Proportions of APCs were determined by flow cytometry. CD14+ monocytes were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14+ monocytes from healthy controls were analyzed for their migratory and spreading capacity. Results Proportions and absolute numbers of monocytes were significantly increased in RA patients treated with CTLA-4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA-4Ig. Likewise, isolated monocytes from healthy controls revealed a significant reduction in their migratory and spreading activity after preincubation with CTLA-4Ig or anti-CD80 and anti-CD86 antibodies. Conclusion We describe direct effects of CTLA-4Ig therapy on phenotype and functional characteristics of monocytes in RA patients that might interfere with the migration of monocytes to the synovial tissue. This additional mechanism of CTLA-4Ig might contribute to the beneficial effects of CTLA-4Ig treatment in RA patients.

While evidence implicates both the adaptive and innate immune system in rheumatoid arthritis (RA) pathogenesis, accumulating data indicate that the synovial tissue itself actively participates in the destructive inflammatory process. Specifically, resident fibroblast-like synoviocytes (FLS), together with macrophages, re-organise to form an aggressive cell mass, called pannus, which destroys the articular cartilage and the subchondral bone. The exact molecular mechanisms of synovial pannus formation, FLS expansion and invasion into adjacent tissues are not yet known. Our data strongly suggest that the T-cell derived cytokine IFNγ is involved in FLS-mediated joint destruction. Migration and invasion assays revealed increased migratory activity for IFNγ-stimulated FLS, when compared to unstimulated FLS. Further, biochemical studies showed that IFNγ promotes the migratory and invasive activity of FLS via Janus kinase 2 (JAK2) and the focal adhesion kinase (FAK), a kinase known to integrate focal adhesion turnover and thus, regulates cell migration. In detail, IFNγ stimulation of FLS distinctly resulted in the phosphorylation of FAK-Y925, a phospho-site that has recently been demonstrated to be required for FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated the IFNγ-induced activation of FAK. Correspondingly, baricitinib, a JAK inhibitor that is currently successfully probed in RA clinical trials, abrogated IFNγ-stimulated activation of FAK. In conclusion, our studies contribute insight into the synovial response to IFNγ and reveal JAK2 and FAK as potential targets for synoviocyte-mediated joint destruction in arthritis, especially in RA.

Background and objectives Fibroblast like Synoviocytes (FLS) are key for the formation of the synovial lining / sublining architecture via unique cadherin-11 mediated cell-to-cell adherens junctions. Binding to the juxta-membrane domain of the cytoplasmic tail of cadherins, p120 is indispensable for the stabilisation of cadherins on the cell surface. By contrast, unbound p120-catenin interferes with small Rho GTPases, thereby regulating actin cytoskeletal organisation as well as NF-κB pathway activity. Here we probe into the function of p120-catenin for synovial tissue formation. Methods Primary FLS were cultured from synovial tissue after synovectomy of RA classified patients. The knock down (KD) of p120-catenin was achieved by the expression of specific shRNA via a lentiviral vector. Immunoblotting was performed to verify decreased p120-catenin expression levels as well as the effects on the expression of cellular cadherin-11. 2D FLS cultures were analysed using phase contrast microscopy as well as confocal immunofluorescence imaging of actin filaments and cadherin-11 mediated cell-to-cell junctions. For the analysis of FLS capacity in forming synovial-like tissue, FLS were cultured in Matrigel® micromasses. Data quantification was done with ImageJ; for statistical analyses Graphpad Prism® was used. Results p120-catenin silenced FLS demonstrated an altered cellular phenotype regarding the size, shape and the cytoskeletal organisation when compared to control cells. In conventional 2D cultures, the surface area covered by KD cells was increased threefold (p Conclusion Although the immunological role of p120-catenin for FLS function in synovial inflammation (NF-kB activity) has yet to be shown, our findings strongly suggest the importance of p120-catenin for the formation of the normal synovial tissue.

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.

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.

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.

Polymorphonuclear neutrophils (PMNs) participate in the initiation of rheumatoid arthritis (RA), but their precise role at the earliest stages of the synovial inflammatory response is only partially understood. We therefore analysed the initial steps of synovial inflammation in the mouse model of collagen-induced arthritis (CIA) with regard to the role of PMN. CIA was induced in C57/BL6 mice. Mice were tested for anti-CII serum antibodies by ELISA. Mice were killed on days 10 and 20 after the first immunisation before clinical …

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 behaviour has not been analysed so far. We analysed 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 characterised 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 behaviour. During phase II (day 3-7) 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. Conclusion The 3D synovial tissue culture system allows to monitor and analysed subtle migration patterns of Mo in relation to the organised 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.

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

In rheumatoid arthritis (RA), a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass, called pannus, which invades into and destroys the articular cartilage. Cartilage destruction is primarily mediated by fibroblast-like synoviocytes (FLS). Among the pro-inflammatory mediators produced by infiltrating T cells, interferon γ (IFNγ) may contribute to FLS driven joint destruction. Of note, IFNγ elicits its effects via the JAK1/JAK2-Stat1 signaling pathway. To establish a role for IFNγ in the invasive potential of FLS, the authors used an in-vitro invasion assay (matrix-associated trans-epithelial resistance invasion-(MATRIN)-assay). Strikingly, FLS that were stimulated with IFNγ demonstrated a markedly increased invasive capacity when compared to un-stimulated FLS. Cell invasion involves several steps, including attachment to extracellular matrix (ECM), cell migration, and digestion of the ECM by proteases. As determined by qPCR, however, IFNγ did not up-regulate the expression of metalloproteinases (MMPs) by FLS. Therefore, the authors hypothesised that IFNγ directs FLS motility. Indeed, exposure of FLS to IFNγ resulted in their increased migratory activity as determined in Boyden chamber assays. Since cell motility is partly controlled by focal adhesion kinase (FAK), the authors next analysed whether or not IFNγ modulates FAK activity in FLS. Western blot analysis revealed that activation of the IFNγ-JAK1/2-Stat1 signaling cascade is associated with phosphorylation of FAK. As Stat1 deficient U3A cells similarly responded to IFNγ stimulation, activation of FAK was independent of Stat1. Instead, inhibition of JAK1/2 abrogated IFNγ induced activation of FAK in FLS, indicating that IFNγ regulates FAK activity through JAK1/2, but not Stat1. Importantly, inhibition of JAKs abrogated IFNγ induced invasive activity of FLS. These results confirm that JAK1/2 play a critical role in translating the signal induced by IFNγ to promote invasion in FLS. These studies suggest a role for IFNγ in the destructive mesenchymal tissue response to inflammation and may provide insight into FLS behavior and function in RA.

Background Granulomonocytic cells (GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely understood. This tempted us to study GMC in the murine collagen induced arthritis (CIA) model of RA with the help of multi-photon real time in vivo microscopy together with the subsequent and sequential ex vivo analysis of GMC on tissue sections. Methods CIA was induced in LysM-EGFP C57BL/6 transgenic animals that carry the EGFP fluorescence protein under the lysozyme promoter. Individual joints were prepared by surgical microscopy in healthy control and in CIA subjects and EGFP + GMC were analysed by 2-photon laser microscopy over 2 h. One group of animals received one single dose (0.25 mg) of prednisolone intravenously before in vivo imaging. Afterwards, the animals were killed and cryo-, and paraffin sections were prepared for immunofluorescence and histomorphological analysis, respectively. Results GMC were barely detectable in healthy animals but were abundant in the synovial tissue as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue with a reduced mean velocity (2.75±1.17 µm/min) of as compared to healthy controls (3.11±1.51 µm/min; p high neutrophilic granulocytes and EGFP low monocytes. In addition EGFPl low F4/80 + TRAP + osteoclast precursor cells were occasionally observed at the synovial-bone junction and areas of bone erosions.Prednisolone treatment reduced the mean velocity of cell migration (2.19±1.06 µm/min; p Conclusion The combined application of real time in vivo microscopy together with elaborate static postmortem analysis of GMC enabled the description of dynamic migratory characteristics of GMC together with their precise allocation in a complex anatomical environment. Moreover, this approach was found sensitive enough to detect subtle therapeutic effects within a very short period of time.

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. In RA, a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass called pannus which attaches to, encroaches over and destroys the articular cartilage. Cartilage destruction is mediated by fibroblast-like synoviocytes (FLS) which are the prevailing cell type of the …