Background Activated macrophages and synovial fibroblasts (SF) are found in the inflamed and hyperplastic synovial rheumatoid arthritis (RA) tissues. Our group has already reported a persistent global hypomethylation in RA tissues and RA synovial fibroblasts (RASF). Recent findings showed that 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 which may explain the genomic hypomethylation in RA. It is known that TET enzymes require for their activation a-ketoglutarate (a-KG) which is produced from isocitrate during cell glycolysis by the enzyme Isocitrate dehydrogenase 1 (IDH-1).
Objectives In the current study, we investigated the 5-hmC modification, TET1–3 and IDH-1 enzymes in RASF and monocyte derived macrophages stimulated with TNFa and LPS.
Methods RASF cultures were prepared from joint replacement patients. The THP-1 cell line was differentiated into macrophages in the presence of 50nM phorbol myristate acetate (PMA). Macrophages were prepared by culturing CD14+ monocytes with macrophage colony stimulating factor (M-CSF) for 6 days. Macrophages and RASFs were transfected with siRNA for TET1 and stimulated with 100 ng/ml LPS for 2 hours or 10 ng/ml TNFa for 24 hours. Glucose uptake was measured using a colorimetric assay. The mRNA levels of TETs and IDH-1 were analyzed by quantitative Real-time PCR. The protein levels of TNFa and IL-6 were measured by ELISA. The global 5-hmC levels were determined by DNA dot blot assay. Hydroxymethylated DNA immunoprecipitation (hMeDIP) was applied to analyze the levels of 5-hmC in different promoter areas of the TNFa (TNFa 1–4) and the IL-6 locus.
Results First, we screened different SF cultures and macrophages for the expression of 5-hmC using dot-blot assay and found that RASF cultures do not express 5-hmC in comparison to CD68+ macrophages. TNFa reduced the expression of TET1 and TET2 mRNA in RASF by 50% and 20% respectively. Interestingly, we observed a 40% reduction of IDH1 (n=3) and an increase of glucose uptake in RASF (n=2). Stimulation of the macrophages with LPS for 0.5h showed a significant increase of TET1 mRNA (TET1: 0.5h 1.93±0.7, fold change, n=4, p=0.03). Interestingly, LPS stimulation increased 5-hmC levels in the promoter of TNFa and IL-6 in monocyte derived macrophages (hMeDIP: TNFa 1.64±0.2, n=3, p=0.03; IL-6 1.74±0.2, n=3, p=0.03). Next, we knocked down TET1 with siRNA in THP1 differentiated macrophages and found that TNFa and IL-6 production was reduced by 46% and 44% as well as the 5-hmC levels were reduced by 25% respectively (n=4).
Conclusions For the first time, we showed that TNFa reduces the glycolytic and epigenetic enzymes IDH1 and TET1 in RASF. In addition, we found that TET1 contributes to the activation of macrophages through the regulation of 5-hydroxymethylation in the promoter of pro-inflammatory cytokine genes. The IDH1 and TET1 enzymes could be promising molecular targets of RASF activation and macrophage chronic inflammation.
Acknowledgement IMI BTCure, EURO-TEAM, IAR Epalinges, FCS scholarship
Disclosure of Interest None declared