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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

Background The synovial membrane is a distinctly organised tissue structure. During the course of rheumatoid arthritis (RA), the synovium becomes hyperplastic and demonstrates thickening of the lining layer and cellular condensation at the sublining layer. Using a three-dimensional synovial organ culture system, we explore cellular mechanisms of synovial tissue formation and inflammatory remodelling. Materials and methods Fibroblast-like synoviocytes (FLS) derived from patients with RA were cultured in 3D micromasses. To mimic synovial inflammation, micromasses were challenged with TNF. For histological analyses, micromasses were embedded in paraffin, sections were stained with haematoxylin and eosin; Ki67 labelling was performed to identify proliferating cells. 3D confocal micrographs were analysed using Imaris Bitplane software. mRNA levels for various genes expressed in FLS were determined by qPCR. Results Synovial micromasses demonstrated thickening of the lining layer over time. When stimulated with TNF, hyperplasia of the lining layer and cellular aggregation at the sublining layer was observed. In order to identify the origin of cells contributing to the thickening of the lining layer, proliferation studies were conducted. Intriguingly, in the early phase of the culture period, the percentage of proliferating cells in the lining layer was higher when compared to the sublining layer. This proliferative activity, however, was no longer present in the late phase, after the lining layer was established. In the presence of TNF, an increased number of proliferating cells at the lining layer was maintained for an extended period of time, consistent with higher rates of cellular proliferation at the synovial lining in sections of RA synovial tissues when compared to OA. qPCR data indicate that MMP1, MMP3, and IL-6 are differentially expressed during the early phase and the mature phase of the culture period. By contrast, lubricin, cadherin-11, CCL20, and STAT1 were not differentially expressed. Conclusions The three-dimensional FLS micromass culture reveals spontaneous cellular organisation that strikingly resembles the lining/sublining architecture of the synovium. This process involves FLS proliferation as well as expression of genes that allow for tissue remodelling. In inflammatory conditions similar cellular programs are re-activated resulting in synovial lining hyperplasia and a pannus-like condensed mass of cells.