Characterization of 3(3,4-dihydroxy-phenyl) propionic acid as a novel microbiome-derived epigenetic modifier in attenuation of immune inflammatory response in human monocytes

Abstract

Recent studies suggest that microbiome derived 3(3,4-dihydroxy-phenyl) propionic acid (DHCA) attenuates IL-6 cytokine production through downregulation of the epigenetic modifier DNA Methyltransferase 1 (DNMT1) expression and inhibition of DNA methylation at the 5'-C-phosphate-G-3' (CpG)-rich IL6 sequence introns 1 and 3 in a mouse model of depression. In this study, we extended the investigation of DHCA epigenetic mechanisms in IL-6 expression in human peripheral blood mononuclear cells (PBMC). Using Lucia Luciferase reporter gene system we identified CpG-rich sequences in which of methylation is influenced by DHCA similar to what observed in response to treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. Correlation study showed that DNA methylation at select CpG motifs in the IL-6 promoter correlates with IL-6 gene expression. Our study suggests that DHCA is effective in reducing IL-6 expression in human PBMCs, in part, by regulation of methylation in the IL-6 promoter region.

Keywords: 3(3,4-dihydroxy-phenyl) propionic acid (DHCA); Epigenetics; IL-6; Inflammation; PBMC; Polyphenols.

Fig. 1. Role of DHCA on LPS-induced IL-6 expression.

The effect of LPS and DHCA on IL 6 gene expression in PBMCs isolated from six age-matched, male healthy donors. The numbers expressed as percentage of individual controls.

Fig. 2. Role DHCA on methylation of CpG dinucleotides of IL-6 promoter.

DNA methylation status of the nineteen CpG dinucleotides in healthy human PBMC samples treated with vehicle control, LPS or LPS and DHCA. Data are shown as mean ± SEM, N=6, in triplicate.

Fig. 3. Human IL-6 promoter region and CpG motifs.

The nineteen CpG dinucleotides in the IL-6 promoter region are highlighted in red. Sequences P-1165/−965. P-697/−394, P-366/−95 enriched in CpG motifs that were cloned into the CpG-free Lucia plasmid for gene expression are highlighted in yellow.

Fig. 4. Examination of methylation of GpC-enriched motifs in IL-6 promoter region and the role of HDAC.

(A) Lucia CpG-free basic plasmid. (B) Luciferase activity in N2a cells transfected with CpG-free basic plasmids with CpG enriched sequences from IL-6 promoter P-1165/−965, P-697/−394 and P-366/−95. (C-E) Luciferase activity in the presence of vehicle, AZA and DHCA following in cells transfected with control and plasmid containing P-697/−394 and P-366/−95 sequences. Data are shown as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001. n=3–5 per assays in duplicate