Background/Purpose:

Activated macrophages are found in the inflamed and hyperplastic synovial RA tissue. Macrophages are the main producers of high levels of pro-inflammatory cytokines such as TNFa and IL6. However, the mechanisms of TNFa overexpression are still unknown. 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. In the current study, we investigated the 5-hmC modification in 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). THP-1 derived macrophages were stimulated with 10 ng/ml LPS at different time points (0h, 2h, 4h, 6h, 24h). The global 5-hmC levels were quantified by DNA dot blot assay. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation assays for TET1 were applied to analyze the levels of 5-hmC in the TNFa promoter. THP-1 derived macrophages were transfected with TET1,2,3 siRNA and then stimulated with 10ng/ml LPS for 2 hours. TNFa and IL6 levels were measured in the supernatants by ELISA.

Results:

The undifferentiated monocytes (THP-1) had the lowest basal levels of 5-hmC. The highest levels of 5-hmC was observed after 24 hours of PMA stimulation after which the monocytes had differentiated into macrophages. Furthermore, we searched for changes in 5-hmC at the promoter of TNFa and found a significant increase in THP-1 derived macrophages (n=3, p<0.05).

THP-1 derived macrophages were stronger responders to LPS than the monocytes for all inflammatory cytokines tested. For each stimulation time point, we measured the 5-hmC modification by hMeDIP and found a time depend increase in the level of 5hmC at the TNFa promoter. The 5-hmC enrichment of the TNFa promoter was significantly higher in THP-1 derived macrophages than THP-1 monocytes (n=3, p<0.05).

Then, we knocked down TET1 in THP-1 derived macrophages before stimulating the cells with LPS. Most importantly, TET1 inhibition reduced the stimulatory capacity of macrophages as shown by the significantly reduced mRNA and protein levels of pro-inflammatory cytokines (TNFa, IL-6, n=3, p<0.05). In contrast, siTET2 and siTET3 did not reduce the expression levels of TNFa and IL-6.

Since the function of TET1 enzymes is to hydroxylmethylate the cytosine residues in gene enhancers and promoters, we measured the levels of 5-hmC at the promoter of TNFa and IL6 by hMeDIP. siTET1 transfected macrophages had significantly lower 5-hmC enrichment in comparison to control cells upon LPS stimulation (n=3, p<0.05). Furthermore, we found direct binding of TET1 at the promoter of TNFa by TET1 ChIP assay (2.5 TET1 fold enrichment versus IgG control, n=3).

Conclusion:

For the first time, we showed that TET1 contributes to the sustained activation of macrophages through the regulation of 5-hydroxymethylation in the promoter of TNFa. The TET1 enzyme could be a promising target to study in macrophages from RA patients.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/tet1-is-an-important-transcriptional-activator-of-the-tnfa-locus-in-macrophages/