Background/Purpose

Lymph node stromal cells (LNSC) build the scaffold that enables migration and interaction of lymphocytes in the lymph node. More recently, it has been shown that during inflammation LNSC play a crucial role in shaping the immune response and maintaining tolerance. Since DNA methylation changes have been reported in autoimmune diseases, we analysed the DNA methylation profile of LNSC from healthy individuals and rheumatoid arthritis (RA) patients.

Methods

Needle biopsies of inguinal lymph nodes were strained through a 70µm nylon mesh and the resulting stromal part was cultured in DMEM with 10% FCS. Adherent cells were passaged 4 times before total genomic DNA was isolated. DNA samples (healthy n=4 and ACPA+ RA n=5) were subjected to the Illumina HumanMethylation 450 array, which allows the analysis of 485.000 methylation sites per sample. It covers the promoters, 5-UTR, 3-UTR, gene body, first exon of 99% RefSeq genes and 96% of CpG islands.  After strict quality control analyses we calculated the differential methylated regions using the COHCAP bioinformatics package in R (version 3.0.1). The results obtained by array analyses were validated for a selected number of probe sequences by pyrosequencing.

Results

The bioinformatics analysis between healthy and RA LNSC revealed 557 significantly differential methylated CpG sites (delta β-value >0.25, p<0.05. We found 57% of the differentially methylated CpG sites to be hypomethylated and 43% hypermethylated.  374 genes were found to be associated with the differential methylated sites. Functional annotation clustering was performed using the hypermethylated (167 genes) and hypomethylated (207) genes. For the hypermethylated genes, the highest enrichment was associated with homophylic cell adhesion and positive regulation of cell death pathways. For the hypomethylated genes, pathways associated with cell adhesion, cell projection, regulation of cell growth and cell motion were significantly differentially methylated. Next, we analysed for specific genes with differentially methylated CpG islands. Interestingly, we identified the hypermethylated genes CXCL4 (RA   β-value=0.46 - healthy β-value=0.20, p=0.008) and Lactotransferin (RA β-value=0.52 – healthy β-value=0.25 p=0.016) that have previously associated with RA. In addition, other gene targets were found to be strongly hypomethylated such as NTGN1 (RA   β-value =0.13 - healthy β-value=0.40, p=0.016) and KCNE1 (RA β-value=0.09-healthy β-value=0.35, p=0.010).