Methylation of the Vitamin D Receptor (VDR) Gene, Together with Genetic Variation, Race, and Environment Influence the Signaling Efficacy of the Toll-Like Receptor 2/1-VDR Pathway

Vanessa Meyer, Donovan Sean Saccone, Fidele Tugizimana, Furaha Florence Asani, Tamsyn Jacki Jeffery, Liza Bornman

The disparity in prevalence of infectious diseases across the globe is common knowledge. Vitamin D receptor (VDR)-mediated toll-like receptor (TLR) 2/1 signaling produces antimicrobial peptides, which is critical as a first line of defense in innate immunity. Numerous studies disclosed the independent role of genetic polymorphisms in this pathway, vitamin D status or season and more recently epigenetics, as factors contributing to infectious disease predisposition. Few studies address the interaction between environment, genetics, and epigenetics. Here, we hypothesized that VDR-mediated TLR2/1 signaling is influenced by a combination of environment, epigenetics and genetics, collectively influencing differential innate immunity. Healthy Black and White South Africans (n = 100) donated blood, while ultraviolet index (UVI) was recorded for the duration of the study. LC-MS/MS supported 25(OH)D3 quantification. Monocyte/macrophage cultures, supplemented with/without 1,25(OH)2D3, were activated with the TLR2/1 elicitor, Pam3CSK4. VDR, cathelicidin antimicrobial peptide, hCAP-18, and 25-hydroxyvitamin D3-24-hydroxylase expression were quantified by RT-qPCR or flow cytometry. Pyrosequencing facilitated VDR methylation analysis and single-nucleotide polymorphism (SNP) genotyping in regions pinpointed through a bioinformatics workflow. Season interacted with race showing 25(OH)D3 deficiency in Blacks. UVI correlated with 25(OH)D3 and VDR methylation, likely influencing race differences in the latter. Regarding the TLR2/1 pathway, race differences in SNP genotype distribution were confirmed and functional analysis of VDR-mediated signaling showed interaction between race, season, and 25(OH)D3 status. Multivariate OPLS-DA mirrored several interactions between UVI, 25(OH)D3 status, DNA sequence, and methylation variants. Methylation of the third cytosine-phosphate-guanine dinucleotide (CpG) in the promoter CpG island (CGI) 1062, CGI 1062 CpG 3, significantly discriminated a 5.7-fold above average mean in VDR protein level upon TLR2/1 elicitation, the variation of which was further influenced by 25(OH)D3 status and the VDR SNP TaqI. Regulation of VDR-mediated TLR2/1 signaling is multifactorial, involving interaction between environment [UVI and consequent 25(OH)D3 status], epigenetics (VDR methylation at key regulatory sites), and genetics (TLR1, TIRAP, and VDR SNPs).