Objective: We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis.Methods: We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically.Results: Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis.Conclusion: AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by synovitis. During inflammation, the synovium transforms into the RA-typical hyperplastic membrane which is composed of fibroblast-like synoviocytes (FLS) and activated resident as well as infiltrating immune cells. By attacking adjacent structures, the hyperplastic membrane drives the destruction of bone and cartilage. Interactions and reciprocal cross-talks between FLS and immune within the synovium promote the pathogenic course of RA. Objectives: Understanding of molecular and cellular mechanisms in disease pathogenesis has led to great advances in the treatment of RA. The role of cell-to-cell interaction in the chronic course of RA however remains largely unknown. In this study, we investigated FLS – immune cell interaction patterns in an inflammatory setting and its consequences on synovial inflammation which will advance clinical biomarker development. Methods: Human FLS were isolated and treated with different inflammatory cytokines to mimic the inflamed joint. After 24h, cytokines were washed off and autologous peripheral blood mononuclear cells (PBMCs) were added to the pre-treated FLS. Automated high-content microscopy of co-cultures after 24h followed by downstream bioinformatic analysis of image data enabled visualisation and quantification of FLS – immune cell interactions. Spectral cytometry analysis of PBMCs re-isolated from co-culture allowed to determine the activation state of PBMC subsets. Sort-purification of PBMC subsets followed by RNA-seq enabled deep phenotyping and profiling of transcriptional changes in monocytes, B-cells, memory CD4 T-cells, and memory CD8 T-cells after co-culture with FLS. Results: Bioinformatic analysis of high-content fluorescence microscopy data showed cell type-specific interaction patterns between FLS and PBMC subsets. Cytokine pre-treatment of FLS had a profound cytokine-specific impact on FLS-immune cell interaction patterns. We also observed enhanced activation of immune cell subsets after co-cultures of FLS and PBMCs. In line with microscopy data, cytokine pre-treatment of FLS modulated their capacity to activate immune cells. IFNγ pre-treatment of FLS increased whereas TGFβ repressed the immune cell activation state. RNA-seq of sort-purified PBMCs after co-culture with FLS revealed a pro-inflammatory phenotype induced in monocytes by IFNγ pre-treatment of FLS. We integrated in vitro generated monocyte signatures of the IFNγ condition into synovial biopsy-derived inflammatory monocyte transcriptomic data which revealed a large overlap of activated genes. Conclusion: This research highlights the important role of FLS in orchestrating synovial inflammation via their interaction with immune cells and further underlines the importance of the synovial cytokine milieu in shaping immune cell development in RA. It further shows the potential of our 2D co-culture model system as a clinical biomarker. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Mirjam Dellinger: None declared, Anela Tosevska: None declared, Felix Kartnig: None declared, Teresa Preglej: None declared, Leonhard Heinz: None declared, Marie Brinkmann: None declared, Bianca Luckerbauer: None declared, Günter Steiner: None declared, Daniel Aletaha AbbVie, Amgen, Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, AbbVie, Amgen, Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, Myles Lewis: None declared, Costantino Pitzalis: None declared, Thomas Karonitsch: None declared, Michael Bonelli Eli-Lilly, Galapagos

Introduction Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. Methods Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4 + T cells from patients with RA was established to quantify cell–cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell–cell interaction. Results Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4 + T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. Conclusion Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

OBJECTIVES:TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS:TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS:TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1β were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION:Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.

Background Rheumatoid Arthritis (RA) is a chronic inflammatory disease, which is characterized by synovial inflammation resulting in bone and cartilage destruction. Crosstalk between activated fibroblast-like synoviocytes (FLS) and immune cells, such as CD4 + T cells, within the synovium might amplify synovial inflammation and joint destruction. Objectives To define the interaction profile of activated FLS and CD4 + T cells within an inflammatory setting and to elucidate its consequence on synovial inflammation. Methods To screen for factors that activate FLS in RA, isolated FLS were treated with different inflammatory cytokines and transcriptomic changes were measured with RNA-seq. Fluorescence activated cell sorting (FACS) purified naïve CD4 + T-cells from the same patients were co-cultured with the cytokine pre-treated FLS. Automated fluorescence microscopy and downstream bioinformatic image analysis allowed visualization and quantification of cell-cell interactions. After co-culture T-cells were isolated and T-cell activation, proliferation and differentiation was determined by flow cytometry. Results To model the in vivo situation, FLS were pre-stimulated with different pro- and anti-inflammatory cytokines. RNA-seq revealed cytokine specific activation patterns of FLS. Correspondingly, we observed distinct CD4 + T cells – FLS interaction profiles depending on the cytokine used for FLS activation. In line with distinct interaction profiles, specific patterns in CD4 + T cells activation, proliferation and differentiation of naïve T cells into CD62L high CD45RO high memory T cells could be detected. Signatures of cytokine-stimulated FLS could be identified in transcriptomic data from synovial tissue samples. Conclusion Within this study, we describe how cytokine induced CD4 + T cells – FLS interactions impact on T-cell proliferation, activation and differentiation. References [1]Zhang F, Wei K, Slowikowski K, Fonseka CY, Rao DA, Kelly S, et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat Immunol. 2019. [2]Smolen JS, Aletaha D, Barton A, Burmester GR, Emery P, Firestein GS, et al. Rheumatoid arthritis. Nat Rev Dis Primers. 2018;4:18001. [3]Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. The Lancet. 2016;388(10055):2023-38. [4]Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233(1):233-55. [5]Zhang F, Wei K, Slowikowski K, Fonseka CY, Rao DA, Kelly S, et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat Immunol. 2019. Disclosure of Interests None declared.