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Rheumatoid Arthritis Patients, Both Newly Diagnosed and Methotrexate Treated, Show More DNA Methylation Differences in CD4+ Memory Than in CD4+ Naïve T Cells


Guderud Kari, Sunde Line H., Flåm Siri T., Mæhlen Marthe T., Mjaavatten Maria D., Lillegraven Siri, Aga Anna-Birgitte, Evenrød Ida M., Norli Ellen S., Andreassen Bettina K., Franzenburg Sören, Franke Andre, Haavardsholm Espen A., Rayner Simon, Gervin Kris

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes pain and swelling of multiple joints in the body. The underlying disease mechanisms are believed to involve a complex interplay between common genetic and environmental factors. The heritability of RA has been estimated to be ~50% for anti-citrullinated protein antibody (ACPA) positive RA and ~20% for ACPA negative RA in a large familial aggregation study (1). Genome-wide association studies (GWAS) have identified more than 100 RA risk loci, mostly conferring risk to ACPA positive RA, marked by lead single nucleotide polymorphisms (SNPs) across various populations (2). The risk SNPs have small effect sizes, and only explain parts of heritability in RA. Environmental and epigenetic factors are also thought to be involved in the RA disease pathogenesis (3) of which smoking is the only established environmental risk factor (4, 5). Epigenetic modifications are important for regulation and maintenance of cell type specific biological functions, and alterations in the epigenome have been found to be associated with RA (6). The most studied epigenetic modification in humans is DNA methylation of cytosine followed by a guanine at so-called CpG sites (CpGs). CpGs are often clustered in regions called CpG islands (CGIs), which frequently overlap gene promoters (7). DNA methylation in promotor regions is usually negatively correlated with transcription of the nearby gene (8). A wide range of immune cells has been implicated in the pathogenesis of RA. One of the most widely used drugs for treatment of RA, methotrexate (MTX) (9), acts as an immunosuppressant in proliferating cells (10), and of these, the most relevant cell population for RA is CD4+ T cells (11). Interestingly, the RA risk loci are enriched in accessible chromatin regions (H3K4me3 peaks) in T cells, including both CD4+ naïve and CD4+ memory T cells (2). Studies have identified cell type specific DNA methylation differences in B (CD19+) and T (CD3+) lymphocytes (12, 13), as well as CD4+ T cells subsets (14, 15) isolated from RA patients compared to healthy controls. However, memory and naïve CD4+ T cells also display distinct genome-wide and gene-specific DNA methylation patterns as a result of normal differentiation (16); hence analyses of bulk T cells may be confounded by different proportions of naïve and memory T cells. Given the recent observations that CD4+ T cell subset distributions are abnormal both in treatment naïve RA patients and in RA patients who has undergone MTX treatment (17) methylation profiles for distinct CD4+ T cell subpopulations should be investigated separately. Methylation levels have so far only been assessed by array-based methods in RA, however reduced representation bisulfite sequencing (RRBS) using next generation sequencers allows for an interrogation of even more CpG sites. RRBS enriches for CpG dinucleotides by utilizes the restriction enzyme MspI (C∧CGG) to digest the DNA sample before bisulfite conversion and sequencing. In this study, we aimed to investigate whether we could detect DNA methylation differences in primary naïve and memory CD4+ T cells from RA patients. To do this, we conducted an epigenome-wide association study using RRBS on isolated T cell populations from two different RA cohorts; (1) disease modifying anti-rheumatic drug (DMARD) naïve RA patients with active disease and (2) MTX-treated RA patients who had been in remission for >12 months. The two cohorts were compared to matched healthy controls.

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Published
February, 2020

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