Background/Purpose: Synovial fibroblasts are key players in rheumatoid arthritis (RA) where they secrete inflammatory cytokines and directly instigate cartilage and bone destruction. However, most treatments are directed at lymphocyte populations, while none directly target these fibroblasts. Here, we report that non-canonical Wnt signaling drives a strong inflammatory gene expression signature among synovial fibroblasts that is associated with R-spondin 3 (RSPO3) and Dickkopf-related protein 3 (DKK3) expression gradients and correlated with RA and other inflammatory diseases.

Methods: We used single-cell and bulk RNA sequencing data from the Accelerating Medicines Partnership RA/SLE Network and the Roche Fibroblast Network Consortium to assess Wnt pathway member expression and gradients. We developed Wnt activation signatures using multivariate regression analysis of RNA sequencing data from human RA synovial fibroblasts stimulated in vitro with a combination of Wnt ligands at various doses and timepoints. We performed immunofluorescence on human RA synovial samples derived from clinical biopsy.

Results: In OA and RA synovium, Wnt pathway members are primarily expressed by fibroblasts (Fig. 1A). We investigated the effects of Wnt activation through RNA sequencing of human RA synovial fibroblasts stimulated with Wnt ligands in vitro. We identified genes that show a dose-responsive association with Wnt stimulation (Fig. 1B) and, unexpectedly, we found a striking enrichment of inflammation pathways (Fig. 1C). Furthermore, we identified an axis defined by reciprocal gradients of expression of RSPO3 and DKK3 that is orthogonal to the Notch-mediated lining-sublining gradient (Fig. 1D). We utilized our in vitro Wnt stimulation results to generate a Wnt enrichment score and found that expression of the positive Wnt regulator RSPO3 is associated with enhanced non-canonical Wnt pathway scores as well as activation markers (Fig. 1E). Moreover, donor-specific enhancement of Wnt5a and RSPO3 signatures was associated with decreased enrichment of signatures for DKK3, a putative Wnt inhibitor (Fig. 1F). We sought to identify the clinical relevance of this transcriptional gradient and validated the divergent protein expression of RSPO3 and DKK3 in synovial fibroblasts using multispectral immunofluorescence of human RA synovial biopsies (Fig. 2A). In synovial fibroblast bulk RNA sequencing data, RSPO3 signatures are correlated with Wnt5a signatures and show enhancement in active RA compared to OA, while DKK3 signatures are decreased in RA (Fig. 2B and 2C). Furthermore, in fibroblasts from salivary gland, lung, and gut from patients with Sjogren’s disease, interstitial lung disease (ILD), and inflammatory bowel disease (IBD) compared with controls, RSPO3 and DKK3 show distinct expression patterns (Fig. 2D) with an association of Wnt5a and RSPO3 signatures in inflamed gut tissue (Fig. 2E).

Conclusion: We demonstrate that a Wnt-associated transcriptional gradient defined by Wnt modulators RSPO3 and DKK3 represents a novel mechanism associated with RA fibroblast inflammatory pathology. This work builds on existing groundwork for a class of stromal-targeted therapeutics for RA and other inflammatory diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/wnt-pathway-regulators-r-spondin-3-and-dickkopf-related-protein-3-demarcate-a-transcriptional-gradient-that-drives-synovial-fibroblast-inflammatory-pathology-in-rheumatoid-arthritis/