Synovial fibroblasts are mesenchymal cells in the synovium that regulate tissue homeostasis in healthy joints. However, in rheumatoid arthritis (RA), synovial fibroblasts assume pathological functions as they recruit infiltrating immune cells that degrade cartilage and bone, leading to joint damage. Therapies aimed at synovial fibroblasts in RA have the theoretical potential to prevent joint damage while sparing side-effects from immunosuppression. However, incomplete understanding of synovial fibroblast heterogeneity and the pathways that regulate their identity pose major challenges to the therapeutic targeting of these cells. We sought to attack this problem by applying cutting-edge single cell technology to examine the biology of synovial fibroblasts in RA.
In this study, we performed single cell RNA-sequencing (scRNA-seq) on over 60,000 synovial fibroblasts and endothelial cells from RA and osteoarthritis patients. By analyzing gene expression profiles of synovial fibroblasts at a single cell level, we found that a gene expression pattern reflects the cellular location of fibroblasts within synovial tissue (Figure). This positional transcriptional program, or positional identity, is partially regulated by vascular endothelium-derived Notch signaling. Using a novel organoid culture system to model synovial tissue formation, we demonstrated that endothelial cells induce fibroblast differentiation and re-create fibroblast positional identity in a Notch3-dependent manner.
We next examined the role of Notch3 in arthritis using a serum-transfer model of murine inflammatory arthritis. To our surprise, mice lacking Notch3 proved resistant to the development of inflammatory arthritis; moreover, arthritis was attenuated by antibody-mediated Notch3 blockade. These results indicate that Notch3 signaling is crucial for synovial fibroblast differentiation during inflammatory arthritis.
The transcriptional identity of synovial fibroblasts is determined based on their location within synovium, a process regulated by Notch3 signaling. Our study provides a molecular mechanism by which synovial fibroblast heterogeneity is created and shows Notch 3 to be a putative therapeutic target. Future work will focus on identifying additional molecular pathways that control synovial fibroblast differentiation in RA.