Background/Purpose:

Activated macrophages are found in the inflamed and hyperplasic synovial RA tissues. Macrophages are the main producers of high levels of pro-inflammatory cytokines such as TNF-a and IL-6. 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. In the current study, we investigated the 5-hmC modification in monocyte derived macrophages stimulated with lipopolysaccharide (LPS), and characterized the function of TET1-3 enzymes during inflammation.

Methods:

The leukemic monocytic cell line THP-1 was differentiated into macrophages in the presence of 50nM phorbol myristate acetate (PMA). 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 were analysed by quantitative Real-time PCR. The global 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 different areas of the TNF-a  (TNF-a 1-4) and IL-6 promoter. THP-1 derived macrophages were transfected with TET1 siRNA and then stimulated with 100ng/ml LPS. TNF-a and IL-6 levels were measured in the supernatants by ELISA.

Results:

Global 5-hmC levels were significantly up regulated during differentiation from THP-1 cells to macrophages (Dot blot: ratio to ssDNA 0h 0.59, 48h 4.13 AU). After 48 hours of differentiation, we observed an increase of 5-hmC enrichments in four different regions of the TNF-a. (hMeDIP: TNF-a1: 2.42, TNF-a2: 2.25, TNF-a3: 2.03, TNF-a4: 2.08, fold enrichment, n=3). 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 TNF-a and IL-6 in primary human macrophages (hMeDIP: TNF-a 1.64±0.2, n=3, p=0.03; IL-6 1.74±0.2, n=3, p=0.03). Since, TET1-3 enzymes catalyze the synthesis of 5-hmC, we knocked down TET1 with siRNA in THP differentiated macrophages and found that TNF-a and IL-6 production was reduced by 46% and 44% respectively (n=4).

Conclusion:

For the first time, we showed that TET1 contributes to the activation of macrophages through the regulation of 5-hydroxymethylation in the promoter of pro-inflammatory cytokine genes. The TET1 enzyme could be a promising therapeutic target to inhibit the persistent inflammation caused by macrophages in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-hydroxylmethylation-regulates-pro-inflammatory-cytokine-expression-in-macrophages/