Rheumatoid arthritis, a chronic systemic inflammatory disease of the joints, leads to disability, reduced quality of life and premature mortality. The target tissue in rheumatoid arthritis is the synovial membrane of the joints, where immune cell infiltration and activation of local stromal cells (e.g. synovial fibroblasts) are key contributors to chronic synovial inflammation and joint damage. Despite the existence of powerful drugs (e.g. tofacitinib, an inhibitor of the JAK-STAT signaling), many patients do not achieve a sufficient response to treatment. Uncovering the heterogeneity of target tissues and the mechanisms by which disease-essential cell populations respond to drugs can make an important contribution to understanding the therapeutic non-responsiveness. The aim of this master thesis was to investigate the cellular composition of synovial tissue, to identify synovial cell populations critical for JAK-STAT signaling and to increase knowledge of the effects of tofacitinib in rheumatoid arthritis. We used three experimental systems: fresh synovial tissue obtained by ultrasound biopsy, cultured synovial tissue, and cultured fibroblasts isolated from synovial tissue from patients with rheumatoid arthritis. The cellular composition of the synovium, the expression of major components of the JAK-STAT pathway in synovial cell populations and the effects of tofacitinib on synovial cells were analyzed using single-cell RNA sequencing. Tofacitinib-induced changes in gene and protein expression in synovial fibroblasts (stimulated with inflammatory factors activating JAK-STAT signaling) were investigated with enzyme-linked immunosorbent assay and quantitative polymerase chain reaction.
Cellular composition of 16 synovial tissues showed a distribution of 66,878 cells into major cell types (fibroblasts, endothelial cells, macrophages, dendritic and natural killer cells, B and T cells, mural and mast cells, pericytes and to date -with scRNA-sequencing - undetected neutrophils). We demonstrated that under our experimental conditions tofacitinib did not significantly affect the synovial cell composition and gene expression of 19,584 cells in 3 ex vivo cultured synovial tissue explants. Synovial fibroblasts strongly expressed components of the JAK-STAT signaling pathway, in particular JAK1 and STAT3, demonstrating that these cells represent important targets of JAK inhibitors. Inflammatory cytokine stimulation in vitro increased expression of IL-6, IL-8, ISG15, TNFAIP3, JUNB and SOCS3 in synovial fibroblasts. Inflammation-driven expression of IL-6, ISG15, JUNB and SOCS3 was tofacitib-dependent, whereas IL-8 and TNFAIP3 were not repressed with tofacitinib. IL-6 and IL-8 protein secretion was consistent with the gene expression changes in synovial fibroblasts.
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