Role of fibroblastic stromal cells and notch signaling in tissue inflammation in RA and SLE

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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) and lupus nephritis (LN) is still emerging. We identified a highly expanded inflammatory subpopulation of fibroblasts in 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), HLA-DR and production of IL-6 and many chemokines. We hypothesize that these CD90+DR+ fibroblasts are key in driving inflammation directly by secretion of 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 in the sublining compartment that express Notch3. When we clustered fibroblasts from active lupus nephritis with fibroblasts from RA synovium, we identified co-clusters of Notch3+ fibroblasts that also express Jag1 in both diseases. Here, we wish to determine if it is Notch 3 signaling on fibroblasts that specifically drives spatial pattering and sublining fibroblast differentiation. 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 WT fibroblasts. In Aim 2, we determine the location of the CD90+DR+ inflammatory cytokine producing fibroblasts and Notch3+ fibroblasts in the synovium and in the kidney in lupus nephritis and determine which fibroblast population(s) most significantly associate with leukocytes (T, B and macrophage). In Aim 3 we further activate synovial and kidney-derived fibroblast lines with inflammatory cytokines in the presence or absence of Notch ligands. We use flow cytometry, RNA-seq, LDA, and trajectory analysis to compare fibroblast cell states induced in vitro with those found in synovium in RA and kidney in lupus nephritis. Then, we extend the Notch gradient concept from fibroblasts to adjacent leukocytes by determining if fibroblast-derived Notch ligands activate attached T cell in organoids. Finally, in Aim 4, we determine if targeted, conditional deletion of Notch signaling in fibroblasts or targeted conditional deletion of Notch ligands on fibroblasts prevents inflammatory arthritis in mouse models. Together, these studies will advance our knowledge of how fibroblasts differentiate and become drivers of inflammation and pathology in chronically inflamed human tissues, and how they might be targeted therapeutically.