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

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.

Grant Number: 5P01AI148102-05
NIH Institute/Center: NIH
Principal Investigator: Michael Brenner