Research Project
10120757
The role of fibroblasts in end organ fibrosis is well established, but insights into their roles in chronic inflammatory diseases in peripheral tissues like rheumatoid arthritis (RA) is still emerging. We identified a highly expanded inflammatory subpopulation of fibroblasts in the sublining region of RA synovial tissue. It accounts for >50% of all fibroblasts in the synovium in RA, but it is a rare population in osteoarthritis (OA). The expanded population is distinguished by high expression of CD90 (Thy1, a sublining marker) and HLA-DR, and the production of IL-6 and many chemokines. We hypothesize that these CD90+DR+IL-6+ fibroblasts are key in driving inflammation directly by secreting inflammatory factors and indirectly by recruiting and activating leukocytes to maintain chronic inflammation. When analyzing single cell RNA-seq data from the RA/SLE Accelerating Medicines Partnership (AMP) Consortium, we found that markers of lining and sublining fibroblasts in synovium were not absolute ? but instead represented a gradient in gene expression in trajectory analysis. We found that this transcriptional gradient corresponds to an anatomic spatial gradient in the synovium emanating from blood vessels. Our data suggest that Notch signaling is a dominant driver of the gradient starting with fibroblasts around blood vessels and extending to sublining fibroblasts that express Notch3 receptors and Jagged (Jag)1 Notch ligands. Here, we wish to determine if Notch 3 signaling specifically on fibroblasts drives the spatial pattering and the differentiation of sublining fibroblasts. To accomplish this, in Aim 1 we use mixed cell organoids with endothelial tubules and fibroblasts to compare spatial pattering and differentiation of Notch3 deficient compared to control fibroblasts. In Aim 2, we determine the location of the CD90+DR+ inflammatory cytokine producing fibroblasts and Notch3+ fibroblasts in the synovium and determine which fibroblast population(s) most significantly associate with leukocytes (T cells, B cells and macrophages). In Aim 3 we activate synovial fibroblast lines with inflammatory cytokines that are found in RA, in the presence or absence of Notch ligands. We use flow cytometry, RNA-seq, LDA, and trajectory analyses to compare fibroblast cell states induced in vitro with those found in the synovium in RA. Then, we extend the Notch gradient concept from fibroblast differentiation to how fibroblast-derived Notch ligands activate attached T cell in organoids. Finally, in Aim 4, we determine if targeted, conditional disruption of Notch signaling in fibroblasts or targeted conditional deletion of Notch ligands in fibroblasts prevents inflammatory arthritis in mouse models. Together, these studies will advance our knowledge of how fibroblasts differentiate in RA to become drivers of inflammation and pathology in chronically inflamed synovial tissues, and how they might be targeted therapeutically in murine models.
