F.S. Peters, A.M.A. Peeters, L.J. Hofland, M.G.H. Betjes, K. Boer, C.C. Baan
Chair(s): dr. Michiel Betjes, Erasmus MC, Rotterdam
Thursday 10 march 2016
13:00 - 13:30h at Foyer
Parallel session: Postersessies XI - Opgesplitst in 3 tijdblokken en 3 categoriëen (klinisch, basaal, donatie)
DNA methylation is responsible for the control of cell function by regulation of gene expression, high methylation in the promoter region is associated with gene silencing. Methylation changes in immune-related genes can influence the immune response after transplantation. In this study, we first investigated the changes in DNA methylation of the pro-inflammatory cytokine interferon-gamma (IFN-γ) during immune activation and second analyzed the effect of the commonly used immunosuppressant tacrolimus.
Patients, materials and methods:
Pure CD3+ T cells were isolated from total PBMCs by MACS negative selection. These T cells were stimulated for 7 days with anti-CD3/CD28, in combination with 10-6 M 5’-aza-2’-deoxycytidine, a demethylating agent, or with 10 ng/mL tacrolimus. Cells were harvested at day 0, 1, 3, 4, 7 and after bisulfite conversion and subsequent PCR, DNA methylation was quantified on two CpG sites (CpG-54 and CpG-186) in the IFN-γ promoter region using pyrosequencing analysis. Flow cytometry was used to analyze IFN-γ protein production.
Stimulation significantly increased the percentage DNA methylation of the IFN-γ promoter from day 0 to day 4 (CpG-54; median: 52% range: (39%-66%) to 64% (54%-75%) with p=0.003, CpG-186: 42% (31%-52%) to 51% (35%-56%) with p=0.04). Addition of tacrolimus did not significantly change the DNA methylation. 5’-Aza-2’-deoxycytidine, which served as a positive control, lead to a significant decrease in DNA methylation from day 0 to day 4 (CpG-54: 52% (41%-66%) to 31% (23%-36%) with p=0.002, CpG-186: 46% (31%-52%) to 22% (18%-32%) with p=0.0006). As expected IFN-γ protein production was completely blocked in the presence of tacrolimus.
Based on these data we conclude that following immune activation the DNA methylation of IFN-γ significantly increased and that this change occurs within 4 days after stimulation. Suppression of IFN-γ protein production in the presence of tacrolimus is not due to a change in DNA methylation after immune activation. Therapeutic concentrations of the immunosuppressant tacrolimus do not alter the methylation status of IFN-γ during immune activation.