Background/Purpose: In rheumatoid arthritis (RA), synovial fibroblasts secrete inflammatory cytokines, degrade cartilage and mediate bone destruction. We have previously identified three synovial fibroblasts subsets with distinct transcriptomic profiles. However, whether synovial fibroblast subsets exhibit differential effector functions during inflammatory arthritis remains unclear. Here, we sought to determine if fibroblast subsets display unique or shared effector functions using a K/BxN serum transfer arthritis model in mice.

Methods: Synovial tissue was isolated from bilateral knees of normal, non-arthritic mice (n = 25) or mice after K/BxN serum transfer (n = 35). Following enzymatic digestion, synovial fibroblasts were divided into three fibroblast subsets: CD90+, CD90-CAD11+, and CD90-CAD11-. In parallel, we isolated human synovial fibroblast subsets (CD90+C34+, CD90+CD34-, and CD90-) from RA and OA (n=10) synovial tissue. Transcriptomic profiles were generated using 1000 cells from each fibroblast subset by Smart-Seq2. Synovial leukocyte (CD45+) infiltration for each tissue sample was determined by flow cytometry to identify inflamed samples (>45% leukocytes).

Results:

1. CD90+ fibroblast subset corresponds to sublining fibroblasts in mice. Principal component analysis using the top 3000 most variable genes identified distinct transcriptomic profiles among mouse fibroblasts subsets sorted based on expression of CD90 and CAD11. Consistent with synovial lining fibroblasts, lining markers PRG4 and FN1 were significantly upregulated in CD90- fibroblasts (p<9.1e-5 and p<3.84e-6, respectively). In contrast, sublining markers CD34 and MFAP5 were highly expressed by CD90+ fibroblasts. PCA of human synovial fibroblast subsets separates lining from sublining fibroblast subsets, suggesting conserved subsets between human and mouse.
2. Lining and sublining fibroblasts exhibit markedly distinct responses in the setting of serum transfer arthritis. We identified 999, 537, and 122 significantly upregulated genes (P<0.0088) in CD90-CAD11+ lining fibroblasts, CD90-CAD11- lining fibroblasts, and sublining fibroblasts, respectively. While there were many genes that were significantly up-regulated by both CD90-CAD11+ and CD90-CAD11- subsets (61), there were only a few that were up-regulated by both CD90+ and CD90-CAD11+ (9) as well as CD90+ and CD90-CAD11- (2). Remarkably, no genes were significantly up-regulated by all three fibroblast subsets, suggesting lining and sublining fibroblasts exhibit unique responses to serum transfer induced arthritis.

Conclusion:

Transcriptomic profiling of mouse and human synovial fibroblasts reveals distinct lining and sublining fibroblasts subsets. During inflammatory arthritis, synovial fibroblasts exhibit distinct subset-specific responses to inflammation, suggesting they play different pathologic roles in arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rna-sequencing-of-mouse-and-human-synovial-fibroblasts-reveals-fibroblast-subset-specific-responses-to-inflammation/