Background Macrophages are found in the inflamed and hyperplasic synovial RA tissues. It is already known that they are stably activated and produce high levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFa). In our previous research, we had reported a persistent global hypomethylation in the RA tissues and RASF. Recent findings have shown the 5-methylcytosine (5-mC) modification of DNA can be converted to 5-hydroxymethylcytosine (5-hmC) through the activation of the family of Ten-Eleven-Translocation (TET1-3) enzymes.
Objectives In the current study, we investigated the 5-hmC modification in monocyte derived macrophages stimulated with Lipopolysaccharide (LPS).
Methods The leukemic monocytic cell line THP-1 was differentiated into macrophages in the presence of 50nM phorbol myristate acetate (PMA) after 48 hours. Primary human monocytes were isolated using CD14 magnetic beads from peripheral blood mononuclear cells (PBMCs). Next, macrophages were prepared by culturing the CD14 monocytes with macrophage colony stimulating factor (M-CSF) for 6 days. THP-1 derived macrophages and human primary macrophages were stimulated with 100 ng/ml LPS for 2 hours. At different time points, the mRNA levels of TETs and TNFa were analysed by quantitative Real-time PCR. The 5-hmC levels were quantified by DNA dot blot assay and presented as chemiluminescence intensity in arbitrary units (AU). Hydroxymethylated DNA immunoprecipitation (hMeDIP) was applied to analyse the levels of 5-hmC in four different regions of the TNFa promoter (TNFa1-4).
Results Global 5-hmC levels were significantly up regulated during differentiation of THP-1 cells to macrophages by induction with PMA (Dot blot: ratio to ssDNA 0h 0.59, 8h 0.62, 24h 0.94, 48h 4.13 AU). After 48 hours of differentiation, we observed an increase of 5-hmC enrichments in four different regions of the TNFa promoter in two independent experiments (hMeDIP: TNFa1: 2.68, TNFa2: 2.56, TNFa3: 2.19, TNFa4: 2.14, fold enrichment, n=2). Next, we stimulated the macrophages with LPS. Stimulation of the macrophages with LPS for 0.5h showed a significant increase of TET1 mRNA expression, which decreased after 1h and 2h stimulation (TET1: 0.5h 1.93±0.7, fold change, n=4, p=0.03). However, the TET2 and TET3 mRNA expression remained unchanged. As a macrophage activation control, the mRNA expression of TNFa was increasing up to 2h of LPS stimulation (TNFa: 27±6.9, fold change, n=3, p=0.004). Interestingly, the global levels of 5-hmC were increased under the same stimulatory conditions (Dot blot: ratio to ssDNA 0h 0.31, 0.5h 0.90, 2h 1.45 AU). The increase in TET1 during LPS stimulation correlated with increasing 5-hmC levels in the promoter of TNFa (hMeDIP: TNFa1: 1.39, TNFa2: 1.48, TNFa3: 1.41, TNFa4: 1.34, n=2). In addition, we confirmed our results using human monocyte derived macrophages and found a similar increase of 5-hmC levels in four regions of the TNFa promoter (hMeDIP: TNFa1: 2.25, TNFa2: 3.40, TNFa3: 2.68,TNFa4: 2.63, n=1).
Conclusions It is shown for the first time that 5-hmC and the enzyme TET1 contribute to the activation of macrophages through the regulation of hydroxymethylation in the TNFa promoter.
Acknowledgements IMI BTCure, EURO-TEAM, FCS scholarship
Disclosure of Interest None declared