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Distinct DNA methylation patterns of rheumatoid arthritis peripheral blood and synovial tissue T cells

ABSTRACT: Objective: To study epigenetic patterns in T lymphocytes that accumulate in rheumatoid arthritis (RA) synovium, we characterized DNA methylation of CD3+ T cells in peripheral blood and synovial tissue in RA and osteoarthritis (OA) patients. Methods: Genomic DNA of CD3+ T cells was isolated from RA (n = 8) and OA (n = 5) patients from blood or synovium at the time of arthroplasty using antibodies and magnetic beads. Methylation was measured using Illumina Infinium MethylationEPIC Kit. Differentially methylated loci (DMLs) and genes (DMGs) were identified using Welch’s t-test. Principal component analysis (PCA), hierarchical clustering and pathway analysis were used to determine relationships among groups. Results: Comparing DNA methylation of CD3+ T cells between peripheral blood and synovial tissue within each disease, 4615 and 164 DMLs were identified in RA and OA samples respectively, resulting in 832 and 36 DMGs. PCA showed that methylation differences in T cells were greater based on location (blood vs. synovium) than based on disease (RA vs. OA). Differentially modified pathways were significantly enriched between RA blood and synovial T cells, especially in genes related to complement, integrin cell surface interactions and P53 pathway. The limited number of DMGs identified between OA blood and synovial T cells did not conform to biologic pathways. Conclusion: The patterns of DNA methylation in RA show location-specific differences related to immune pathways, while methylation differences in OA are limited. The RA joint-specific signatures could be due to selective accumulation of T cell populations or expansion of differentially marked adaptive immune cells. Understanding epigenetic patterns could provide clues to the types of T cells that accumulate in the RA joint and identify potential therapeutic targets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164468 | GEO | 2021/02/01

REPOSITORIES: GEO

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Project description:Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response program. We tested the hypothesis that a serum response program can be used to classify diseased tissues, and investigated the serum response program in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including RA, OA and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response program discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through ?3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts. Fibroblasts were isolated from synovium, bone marrow and skin tissue samples taken at the time of knee or hip replacement surgery from 12 rheumatoid arthritis patients meeting the 1987 ACR criteria and 6 osteoarthritis patients diagnosed on the basis of characteristic x-ray findings and the absence of features suggestive of inflammatory arthritis. Only one hip sample was present in either disease group. Fibroblasts were maintained in fibroblast medium (consisting of 81.3% RPMI 1640, 10% FCS, 0.81x MEM non-essential amino acids, 0.81mM sodium orthopyruvate, 1.62mM glutamine, 810U/ml penicillin and 81?g/ml streptomycin) at 37°C in a humidified 5% CO2 atmosphere.

Project description:Purposes Recurrent synovial bleeding is the major cause of Hemophilic Arthritis (HA). Up to now, the transcriptomic profiles of bleeding syvonium in HA patient have largely remained unknown. Exploring the transcriptomic characteristics of synovium obtained from HA patients undergoing total knee arthroplasty (TKA), and uncovering potential pathological mechanisms of HA synovium through bioinformatics analysis and differential analysis. Methods Single-cell RNA sequencing (scRNA-seq) technology was utilized to identify distinct cellular subsets within HA synovium. Comparative analysis was conducted with scRNA-seq data from osteoarthritis (OA) and rheumatoid arthritis (RA) synovium to assess transcriptional differences. Histological evaluation, immunofluorescence (IF), immunohistochemistry (IHC) and in vitro cell testing were performed for validation. Results We observed that the single-cell transcriptomic characteristics of HA synovium differ significantly from those of OA and RA patients. Mast cells, identified as unique immune cells in HA synovium, are actively involved and may be the initiating factor for changes in vascular permeability or bleeding in HA synovium. The transcriptomic features of endothelial cells and fibroblasts, which are non-immune stromal cells, reveal the synovial microenvironment: repeated bleeding leads to iron deposition, which causes substantial ferroptosis within the synovium. Based on the pseudotime analysis of endothelial cells, along with IHC staining and in vitro cell assays, it was demonstrated that ferroptosis in endothelial cells induces vascular damage and triggers a significant migration of endothelial cells. The stress of ferroptosis leads to the differentiation of HMOX1+ endothelial cells. Cell interaction analysis shows that HMOX1+ endothelial cells can induce the SCARA5+ fibroblast subtypes via FTL/SCARA5. This may be a critical target for alleviating cellular ferroptosis in HA synovium. Conclusions Through scRNA-seq, we elucidated the heterogeneity of synovial tissues in three diseases. Activated mast cells around blood vessels may be potential target cells for bleeding and rebleeding, with TPSB2 emerging as a potential target. We also identified endothelial cell subpopulations under ferroptosis stress and their interactions with fibroblasts. Notably, we discovered a SCARA5+ fibroblast subpopulation that regulates ferroptosis, which could serve as a potential target gene for preventing and treating recurrent bleeding in HA.

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Distinct DNA methylation patterns of rheumatoid arthritis peripheral blood and synovial tissue T cells

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