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Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα. Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Here, the authors identified a previously unknown signaling circuit that contributes to tumor necrosis factor (TNF)-induced activation of FLS. The authors show that FOXO3 and its modulator PIK3IP1 are crucial for the TNF-driven interferon (IFN) response in RA-FLS.

Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Introduction The synovium demonstrates a distinct cellular structure with a densely packed synovial lining layer that sits on top of a loosely organised sublining layer. In rheumatoid arthritis (RA), this tissue hosts the inflammatory reaction and undergoes drastic structural changes. We hypothesise that this inflammatory remodelling results in tissue dysfunction, thereby perpetuating the inflammatory process. To explore the relationship between tissue structure and function, we use a 3D human synovial organoid culture system to model healthy and diseased synovium. Methods FLS from RA patients were resuspended in Matrigel and cultured as 3D organoids for an extended period of time. This allows FLS to re-establish a synovial tissue-like structure. To mimic inflammation, 3D synovial organoids were challenged with TNF. A re-stimulation experiment was designed in which synovial organoids were exposed to TNF for 10 days, followed by a 3 day wash out phase. Thereafter, the organoids were re-stimulated with TNF. To prevent TNF-driven remodelling, Marimastat, a broad MMP inhibitor, was added during the first TNF stimulation. For selected experiments, synovial tissue-like structure was assessed by HE staining in paraffin embedded sections of synovial organoids. qPCR and RNA Seq were used in gene expression profiling. IL-6, IL-8 and MMP3 protein production was determined by ELISA. Results TNF-stimulated synovial organoids demonstrated lining layer hyperplasia and cellular condensation in the sublining layer. After TNF-stimulation, gene expression of MMP1, MMP3 and IL-6 and protein production of IL-6, IL-8 and MMP3 was upregulated. Upon re-stimulation of synovial organoids with TNF, protein levels of IL-6, IL-8 and MMP3 were significantly increased when compared to previous stimulations. In order to explore whether this effect is due to TNF-induced structural changes, tissue remodelling was blocked using Marimastat. IL-6 protein level was reduced in 21%. To further explore the increased TNF response upon re-stimulation, epigenetic mechanisms are currently being tested. Conclusions TNF stimulation has a direct effetc in synovial organoids function and structure. TNF-driven tissue remodelling is associated with an increased in IL-6, IL-8 and MMP3 expression response upon re-stimulation. Inhibition of TNF-induced remodelling partially prevents this effect. Additional epigenetic mechanisms may account for tissue memory. Disclosure of interest None declared

Introduction The synovium is primarily built by fibroblast-like-synoviocytes (FLS). In the healthy synovium FLS form a complex tissue network via long-distance intercellular connexions (nanotubes). Using a 3D synovial micromass culture system, our previous research demonstrated that FLS exchange cytoplasmic cargo, i.e. organelles like mitochondria, via transfer through direct cell-to-cell connexions. The adaptive synovial tissue response to inflammation (i.e. to TNFa) likely depends upon the concerted communication between FLS. Objectives To determine how the cellular organisation of the synovial tissue affects intercellular cargo exchange and how it changes under inflammatory conditions. Methods Human FLS were isolated from joint tissues after synovectomies; passaged FLS were used to generate 3D micromass cultures using Matrigel (BD). For ultrastructural 3D reconstruction ultrathin sections of fixed micromasses were cut with an ATUMtom for FE-SEM scanning, followed by alignment and reconstruction of individual cells with Fiji TrackEM. To compare unstimulated and TNF-stimulated micromass cultures, cells were dyed with Cell- and Mitotracker dyes (TF). Using confocal microscopy, micromass cultures were analysed for cellular organisation within the 3D sphere, cell volume of individual cells, cell-to-cell interconnectivity and cargo transfer between cells. Analyses of the 3D confocal imaging data were done in Bitplane Imaris. Results SEM 3D reconstruction of synovial micromass tissue revealed that FLS are compartmentalised, and thus, much larger and extended than expected. Thin membrane tubes not only connect different cells but also compartments of the same cell. Additionally, one nanotube may provide a link to several other cells and these connexions are in general separated by membranes. Treatment of micromasses with TNFa resulted in cellular condensation, reduced cell volume as well as diminished cellular connectivitiy when compared to unstimulated micromasses. In particular, the abundance of interconnecting nanotubes was significantly attenuated. The mitochondrial transfer rate increased as FLS clustered upon TNFa stimulation. Conclusions TNFa directs FLS cellular re-organisation that is associated with increased intercellular transfer of mitochondria. These studies may provide insight into the concerted cooperation of FLS that is likely critical for the adaptive synovial response to inflammation. Disclosure of interest None declared

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA 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, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions 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. Disclosure of Interest None declared

Social learning is considered one of the hallmarks of cognition. Observers learn from demonstrators that a particular behavior pattern leads to a specific consequence or outcome, which may be either positive or negative. In the last few years, social learning has been studied in a variety of taxa including birds and bony fish. To date, there are few studies demonstrating learning processes in cartilaginous fish. Our study shows that the cartilaginous fish freshwater stingrays (Potamotrygon falkneri) are capable of social learning and isolates the processes involved. Using a task that required animals to learn to remove a food reward from a tube, we found that observers needed significantly (P

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.

A taxonomy of the movement possibilities for any species, within the constraints of its neural and skeletal systems, should be one of the foundations of the study of its behaviour. Caribbean reef squid, Sepioteuthis sepioidea, appear to have many degrees of freedom in their movement as they live in a three-dimensional habitat and have no fixed skeleton but rather a muscular hydrostatic one. Within this apparent lack of constraints, there are regularities and patterns of common occurrences that allow this article to describe an ethogram of the movements, postures and positions of squid. Squid have a combination of bent, spread and twisted maintained postures of the eight arms and two tentacles that enhance camouflage. Their body–arm posture combinations are actively maintained in the water but also influenced by gravity. Positions related to conspecifics are stereotyped and important for communication. For locomotion, squid use a well-coordinated dual fin–jet locomotion system. This motor system uses tonic...

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.

Testing the cognitive abilities of cartilaginous fishes is important in understanding the evolutionary origins of cognitive functions in higher vertebrates. We used five South American fresh water stingrays (Potamotrygon castexi) in a learning and problem-solving task. A tube test apparatus was developed to provide a simple but sophisticated procedure for testing cognitive abilities of aquatic animals. All five subjects quickly learned to use water as a tool to extract food from the testing apparatus. The experimental protocol, which gave the animals the opportunity of correcting a wrong visual cue decision, resulted in four out of five subjects correcting an error rather than making an initial right choice. One of five subjects reached 100% correct trials in the visual discrimination task. The ability to use water as an agent to extract food from the testing apparatus is a first indication of tool use in batoid fishes. Performance in the instrumental task of retrieving food from a novel testing apparatus and the rapid learning in the subsequent discrimination/error correction task shows that cartilaginous fish can be used to study the origins of cognitive functions in the vertebrate lineage.

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.

This study aims to investigate the octopus' eye and arm coordination and raises the question if visual guidance determines choice of arm use. Octopuses possess eight seemingly identical arms but have recently been reported to show a preference as to which arm they use to initiate contact with objects. These animals also exhibit lateralized eye use, therefore, a connection between eye and arm preference seems possible. Seven Octopus vulgaris were observed during approach, contact initiation and exploration of plastic objects that were positioned on three different levels in the water column. The subjects most commonly used an arm to initiate contact with an object that was in a direct line between the eye used to look at the object, and the object itself. This indicates that choice of arm use is spatially rather opportunistic when depending on visual guidance. Additionally, first contact with an object was usually established by the central third of the arm and in arm contact sequences neighboring arms were the most likely to follow an arm already touching the object. Although results point towards strong eye/arm coordination, we did not find lateralized behavior in this experiment. Results are discussed from a neuro-anatomical, behavioral and ecological perspective.

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 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

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 …