Background/Purpose: In rheumatoid arthritis (RA), tissue resident fibroblasts orchestrate chronic inflammation and regulate pathologic bone and cartilage remodeling that causes irreversible joint damage. Recently, we and others have identified the selective expansion of CD90+ sublining fibroblasts in RA patients with active, leukocyte-rich synovial histological features. Here, we leveraged droplet-based single cell RNA-sequencing (scRNA-seq), confocal microscopy, and ex vivo tissue organoid cultures to elucidate molecular pathways that govern the expansion of patholgoic synovial fibroblasts in RA.

Methods:

Single cell RNA-seq. Synovial stromal and endothelial cells from RA and OA synovial tissues were isolated and subjected to droplet-based single cell RNA-sequencing. Gene mapping, read alignment, pseudotime and trajectory analyses were performed.

Confocal Microscopy. RA and OA synovial tissue sections were stained with antibodies against (PRG4)/Lubricin, CD90, MCAM (CD146), VWF, and NOTCH3.

3-D synovial organoids. Synovial organoids comprised of fibroblasts and endothelial cells were reconstituted using an in vitro micromass culture system.

Results:

ScRNA-seq of 32,000 single synovial stromal cells identified lining (PRG4+) fibroblasts, sublining (CD90+) fibroblasts, pericytes (ACTA2+), and endothelial cells (VWF+) as the major tissue resident cells in the synovium. Pseudotime and trajectory analysis revealed unexpected fibroblast transcriptional programs that follow anatomical spatial localization. Confocal microscopy visualization of fibroblast markers CD90, Lubricin and MCAM confirmed a spatial gradient along a perivascular to synovial lining axis. Isolation of spatially-restricted fibroblasts followed by serial passages and parallel transcriptomic profiling revealed spatial transcriptomic signatures diminish after serial passages, suggesting spatial gradients is maintained by local signals derived in situ in the synovial microenvironment. Receptor-ligand analysis followed by an in vitro ligand screen identified endothelial-derived Notch signaling as a key driver in establishing the spatial gradient between that perivascular and lining layer fibroblasts. Direct fibroblast-endothelial cell contact using a novel 3-D organoid system recapitulates the in vivo fibroblast-endothelial structure in a Notch-dependent manner. Inhibition of Notch signaling by small molecules and siRNA-mediated silencing of Notch3 abolishes sub-lining formation in vitro. In RA, synovial fibroblasts were overrepresented in the perivascular zone characterized by marked increase in Notch signaling, suggesting Notch regulates pathologic synovial remodeling in RA.

Conclusion:

Using scRNA-seq, we identified a spatial gradient in synovial fibroblasts regulated by endothelium-derived Notch signaling. In RA, Notch signaling drives expansion of sublining (CD90+) synovial fibroblasts. Inhibition of Notch3 signaling prevents CD90+ synovial sublining expansion, highlighting fibroblast Notch3 signaling as a novel therapeutic target in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-rna-sequencing-of-rheumatoid-synovial-fibroblasts-reveals-a-disease-associated-spatial-gradient-modulated-by-inductive-notch-signaling/