Background/Purpose:

Rheumatoid arthritis (RA) is an aggressive immune-mediated joint disease with synovial inflammation and joint destruction. Fibroblast-like synoviocytes (FLS) play a key role in mediating inflammation and joint damage. FLS are usually isolated from enzymatically dispersed synovial tissue and expanded in culture for multiple passages to achieve relatively homogenous population. Epigenetic alteration in RA FLS such as DNA methylation has been demonstrated stable for many passages. One critical question is whether the pathogenic genes altered in later passage FLS reflects the epigenome in situ. Therefore, we studied DNA methylation in the primary (P0) passage synovial fibroblasts (SF) and compared the patterns to late passage cells.

Methods:

Genomic DNA from cultured FLS (30 RA, 16 OA) and P0 SF (5 RA, 10 OA) was isolated from synovial tissues obtained at the time of total joint replacement. P0 SF were isolated by enzymatically disaggregating cells and negatively selecting either using anti-CD45 magnetic beads or adherence to plastic. The P0 cells were evaluated by flow cytometry for T cells (RA 1.1±0.2%, OA 0.4±0.1%), B cells (RA 0.2%, OA 0.1%). Methylation levels were measured using Illumina HumanMethylation450 chip. Differentially methylated loci (DMLs) were identified using Welch’s t-test and mapped to gene promoter regions to define differentially methylated genes (DMGs). The overlapped DMGs were calculated using Hypergeometric test. To compare enriched biological pathways, Ingenuity pathway analysis was applied.

Results:

For P0 SF, 18,537 DMLs (difference of β value > 0.1 and q value < 0.05) were identified in 2,756 DMGs. Compared to previously reported 1,714 DMGs in passage 5 (P5), 582 (34.0%) DMGs of P5 were overlapped with DMGs in P0 (p value = 1.32e-149). 75 differentially methylated pathways were significantly enriched between RA and OA in P0, and 15 of them are overlapped with 52 pathways enriched in P5, such as “Complement System” and “NF-κB Signaling” associated with immunity and inflammation.

Because the limited number of P0 RA samples may be underpowered, we decreased DML stringency to q value cutoff of 0.1 to allow more DMGs and enriched pathways included. As a result, 26,267 DMLs (difference of β value > 0.1 and q value < 0.1) located on 3,578 DMGs are identified between RA and OA FLS in P5, in which 798 (46.6%) DMGs of P5 were overlapped with DMGs in P0 (p value = 1.04e-241). In addition, out of 108 significantly enriched pathways between RA and OA in P0, 19 of them are overlapped with pathways enriched in P5. Interestingly, RA-specific pathway “Role of Osteoblasts, Osteoclasts and Chondrocytes in Rheumatoid Arthritis” is overlapped.

Conclusion:

In this study, we have compared the DNA methylation signatures in RA between P0 SF and classically prepared P5 FLS. Even though there were differences in cell populations and processing methods, the overlap was highly significant with nearly half of DMGs between RA and OA in P5 FLS can be found in the P0 SF. Despite potential limitations due to sample size and the fact that P0 SF includes several subsets of fibroblasts, the DNA methylation signature of classical RA FLS in established culture are consistent with the patterns observed in SF.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stability-of-dna-methylation-signature-in-primary-ra-synovial-fibroblasts-compared-with-cultured-fibroblast-like-synoviocytes/