Researchers have identified a naive B cell signature prior to rheumatoid arthr itis flares, as well as a type of mesenchymal cell, that may play an important role in flare pathways.
But they noticed other patterns as well. “What we weren’t expecting was that we saw certain genes go down during flare, representing things that we wouldn’t have expected blood cells to make,” Dr Orange notes. “It was a little surprising to see in the blood all these things going down that represented pathways of cartilage and bone development.”
To dig deeper, the group used a time-series longitudinal analysis to help identify genetic expression patterns prior to flare. They identified a cluster of genes that became activated two weeks before a flare, which they termed antecedent cluster 2 (A2). They also identified a different set of genes, antecedent cluster 3 (A3), that became highly activated one week before the flare and then were downregulated during the flare itself. Dr. Orange explains, “That group of genes really overlapped with what we had seen previously: There were these cartilage and bone and extracellular matrix genes.”
Single-Cell RNA Sequencing of Synovial Cells & Flow Cytometry Techniques
To try and make sense of these results, the team turned to a previously published data set. Zhang et al. analyzed synovial tissue samples from patients with rheumatoid arthritis or osteoarthritis.3 They used bulk RNA sequencing and single-cell RNA sequencing of individual T cells, B cells, monocytes and fibroblasts. Unlike standard RNA sequencing, which looks at aggregated genetic expression from multiple cells, single-cell RNA sequencing allows scientists to analyze genetic expression in a single, specific cell.
Using this and flow cytometry, which can separate cells into subtypes based on various cell surface proteins, the researchers had identified multiple distinct cell populations, with different patterns of genetic expression and cell surface proteins. These included three distinct types of sublining fibroblasts, which live in the synovial sublining layer of the joint synovium. Some of these sublining fibroblasts are present in greater numbers in the inflamed synovium of patients with rheumatoid arthritis.3
Combining these existing data with their findings, Dr. Orange et al. performed an analysis of the A2 and A3 gene clusters. The A2 cluster was enriched with naive B cell genes. The A3 cluster was heavily enriched with the specific genes that Zhang et al. had demonstrated were prominent in sublining fibroblasts. When they looked at the expression of genes that were common to both these synovial sublining fibroblasts and the A3 gene cluster, they again found increased gene expression one week prior to flare and decreased expression during the flare itself.
All solid tissues have fibroblasts residing in them that are responsible for making and maintaining the tissue scaffolding. Traditionally these cells are defined as being of mesenchymal origin, and they are generally not thought to circulate in the blood. But this is exactly where Dr. Orange et al. had found this genetic signature.
“It has been becoming clear for a while that fibroblasts [in the synovium] in rheumatoid arthritis are quite activated. They express inflammatory cytokines and metalloproteinases and are thought to be playing a pathogenic role in rheumatoid arthritis,” says Dr. Orange. However, no role had previously been established for a fibroblast-like cell in the blood in rheumatoid arthritis pathogenesis.
