Date: Monday, November 9, 2015
Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis II
Session Type: ACR Concurrent Abstract Session
Session Time: 4:30PM-6:00PM
Background/Purpose: Synovial fibroblasts play crucial roles in the pathogenesis of rheumatoid arthritis (RA). They expand as part of the pannus, mediate degradation of cartilage, amplify inflammation, and contribute to osteoclastogenesis. Considering these diverse roles of synovial fibroblasts, we hypothesized that synovial fibroblasts may consist of several subsets with distinct functions, contrary to the common view of fibroblasts as a homogeneous population. Here we sought to identify fibroblast subsets and to characterize subpopulations important in the pathology of RA.
Methods: Synovial cells were isolated by enzymatic digestion from surgical specimens from osteoarthritis (OA) and RA patients. Flow cytometry was used to separate freshly isolated fibroblasts into subpopulations based on the expression of several mesenchymal cell markers. In our initial analysis, nine fibroblast subpopulations were collected by cell sorting and subjected to further analysis by Affymetrix GeneChip Human Gene 2.0 ST microarray. To validate results, we applied low-input RNA-sequencing and single cell RNA-sequencing to independent samples. The functions of these fibroblast subsets were examined by several in vitro assays including the response to TNF stimulation and co-culture with macrophages.
Results: Freshly isolated synovial fibroblasts were divided into several subpopulations based on the expression patterns of fibroblast markers including podoplanin, cadherin11, CD90, CD34, and CD146. The analysis of gene expression microarray data revealed 2,986 genes with significant (1% FDR) variation across fibroblast subpopulations. An independent sample of RA donors, from different joints than the original samples, and assayed via RNA-seq, showed consistent variation of these genes across fibroblast subpopulations. Pathways enriched with this variation included extracellular matrix disassembly (P=7.51e-11) and the TNF signaling pathway (P=1.96e-6). We defined distinct functional cell populations by population-specific expression of genes with well-studied functions. For example, CD34+, CD90+, cadherin11+ cells highly expressed IL-6, CCL2 and CXCL12. CD34-, CD90+, cadherin11+ cells highly expressed TNFSF11. CD34-, CD90- cells highly expressed IL-8, CXCL1, MMP-1 and MMP-3. Differential expression of several genes that mediate distinct fibroblast functions was confirmed at the protein level after culturing cells in the presence or absence of TNF. Functionally, CD34+, CD90+, cadherin11+ cells skewed macrophages into anti-inflammatory phenotype. CD34-, CD90+, cadherin11+ cells promoted osteoclastogenesis. In RA synovial tissue, the proportion of CD34-, CD90+, cadherin11+ cells was increased compared to that in OA.
Conclusion: 1) Synovial fibroblasts are composed of several subsets that are distinct in surface phenotype, transcriptional programs, and functional effects on other cell types. 2) The heterogeneity of fibroblasts and the selective expansion of particular subsets are relevant to understanding RA pathology.
To cite this abstract in AMA style:Mizoguchi F, Slowikowski K, Chang SK, Rao DA, Nguyen H, Noss EH, Earp BE, Blazar PE, Wright J, Simmons BP, Hacohen N, Nigrovic PA, Raychaudhuri S, Brenner MB. Identification of Synovial Fibroblast Subsets That Define Pathology in Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/identification-of-synovial-fibroblast-subsets-that-define-pathology-in-rheumatoid-arthritis/. Accessed October 25, 2021.
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