Regulation Of FcεRIγ From Human Monocytes (MOs) By DNA Methylation And Its Role In Atopic Dermatitis(AD)

Background Environmental factors influencing the manifestation of atopic diseases, which imply epigenetic modification of key genes may contribute to the pathogenesis of atopic disease. The high-affinity receptor for IgE (FcεRI) is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases,FcεRI gamma subunit(FcεRIγ) is key modulator for the maturation and surface expression of FcεRI. In this context, Structural factors influencing the expression of FcεRIγhave gained considerable interest. Recent research revealed that there are two Alu repeats commonly regulated by DNA methylation in the FcεRIγsubunit transcription pomotor, so we investigated the putative role of DNA methylation in the regulation of FcεRIγgene expression in atopic monocytes.Objective To study the putative role of DNA methylation in the regulation of FcεRIγgene and FcεRI expression in human monocytes.Methods PBMCs (peripheral blood mononuclear cells) from 3 healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation, MOs were isolated from the PBMC using magnetic cell separation technique. MOs were cultured in lipopolysaccharide(LPS), then cells were harvested at 72h after addition of 5-azaC. Bisulfite sequencing was used to determine the methylation status of the FcεRIγpromoter region. FcεRIγwas detected by real-time RT-PCR and Western blotting and FcεRI surface expression was detected by flow cytometry. Results Compared with the untreated MOs, treating healthy monocytes with the demethylating agent 5-azacytidine decreased promoter methylation level (P=0.003), and increased FcεRIγand surface FcεRI expression (FcεRIγprotein level:P=0.007, mRNA level:P=0.008, FcεRI surface (%):P=0.001, MFI:P=0.005).Conclusion DNA methylation regulated FcεRIγsubunit gene expression and contributed to the expression of FcεRI on MOs.Objective To investigate the expression levels and methylation patterns of FcεRIγin MOs from AD patients and healthy controls.Methods PBMCs were isolated from the peripheral venous blood of 10 AD patients and 10 healthy donors by density gradient centrifugation, MOs were isolated from the PBMC using CD 14 beads. Bisulfite sequencing was used to determine the methylation status of the FcεRIγpromoter region. Levels of FcεRIγwere measured by real-time RT-PCR and Western blotting, and using the flowcytometry to detect the expression levels of FcεRI in MOs.Results Compared with the healthy controls, the levels of FcεRIγand surface FcεRI expression in monocytes are increased in AD patients (FcεRIγprotein level:P=0.000, mRNA level:P=0.01, FcεRI surface(%): P=0.045, MFI:P=0.000). The DNA methylation levels within FcεRIγregulatory domains are reduced in atopic monocytes relative to healthy controls (P=0.001) and negatively correlate with FcεRIγexpression (R=-0.711,P=0.021).Conclusion DNA hypomethylation leads to FcεRIγsubunit and surface FcεRI overexpression in atopic dermatitis.