Age-dependent VDR peak DNA methylation as a mechanism for latitude-dependent multiple sclerosis risk

Abstract

Background: The mechanisms linking UV radiation and vitamin D exposure to the risk of acquiring the latitude and critical period-dependent autoimmune disease, multiple sclerosis, is unclear. We examined the effect of vitamin D on DNA methylation and DNA methylation at vitamin D receptor binding sites in adult and paediatric myeloid cells. This was accomplished through differentiating CD34+ haematopoietic progenitors into CD14+ mononuclear phagocytes, in the presence and absence of calcitriol.

Results: Few DNA methylation changes occurred in cells treated with calcitriol. However, several VDR-binding sites demonstrated increased DNA methylation in cells of adult origin when compared to cells of paediatric origin. This phenomenon was not observed at other transcription factor binding sites. Genes associated with these sites were enriched for intracellular signalling and cell activation pathways involved in myeloid cell differentiation and adaptive immune system regulation.

Conclusion: These results suggest vitamin D exposure at critical periods during development may contribute to latitude-related differences in autoimmune disease incidence.

Keywords: Calcitriol; DNA methylation; Epigenetics; Myeloid; VDR binding site; Vitamin D.

Fig. 1

Light microscopy and flow cytometric characterisation of cultured cells at day 22. Cultured cells of adult origin with media containing 0 nM calcitriol (left), 0.1 nM calcitriol (centre) and 50 nM calcitriol (right). There were marked morphologic and immunophenotypic changes, with overall decrease in cell number, fewer fusiform-shaped cells, greater CD14+ proportion and decreases in HLA-DR and CD16 expression (not shown) at higher calcitriol concentrations

Fig. 2

Global DNA methylation by age and calcitriol status. a Frequency histograms of 1-kb tile, genome-wide DNA methylation, showing similar distribution of DNA methylation between conditions. b Multidimensional scaling analysis of CpG-wise methylation values demonstrating only minor differences in DNA methylation with the addition of calcitriol. A: adult, P: paediatric, N: no calcitriol, D: with calcitriol, 1 or 2 refer to adult or paediatric subject 1 or 2

Fig. 3

Distribution of DNA methylation by genomic feature. a Violin plots of DNA methylation across various genomic features by cell origin and culture condition. Myeloid VDR peaks demonstrated DNA methylation that was skewed towards lower methylation levels in cells of paediatric origin in comparison to those of adult origin. The effects of calcitriol on DNA methylation distribution was not evident. b Methylation difference between cells of paediatric and adult origin at myeloid VDR peaks and transcription factor binding sites, showing skewing towards paediatric hypomethylation at myeloid VDR peaks, but not at transcription factor binding sites. A: adult, P: paediatric, N: no calcitriol, D: with calcitriol, 1 or 2 refer to adult or paediatric subject 1 or 2

Fig. 4

Characteristics of differentially methylated VDR peaks. a Breakdown of VDR peaks based on differential methylation status and overlap with regions of interest (regulatory regions, CpG islands and island shores; left) and differentially methylated VDR peaks overlapping with regions of interest (right). The majority of regulatory regions demonstrated hypomethylation in cells of paediatric origin. b Overrepresented GO terms (FDR < 0.05) associated with differentially methylated VDR peaks. c Breakdown of differentially methylated myeloid VDR peaks and corresponding annotation overlaps compared with all annotated VDR peaks. d Overlap of currently known non-HLA MS risk genes and their overlap with differentially methylated myeloid VDR peaks (Additional file 5). PTK: protein tyrosine kinase, PI3P: phosphatidylinositol-3-phosphate, CGI: CpG island, PFR: promoter flanking region, TFBS: transcription factor binding site

Fig. 5

Differentially expressed genes, associated gene ontologies and DNA methylation overlap. a Number of genes demonstrating changes in expression (> twofold) due to calcitriol in cells of adult and paediatric origin. b A minority of common genes are differentially expressed in response to calcitriol amongst cells of adult and paediatric origin. c Overlap between differentially expressed genes and differentially methylated myeloid VDR peaks/genes when comparing cells of adult and paediatric origin. d Scatter plot of overlapping sites from c corresponding to annotated promoter regions. There was a significant negative correlation between methylation difference (paediatric–adult) and log fold-change (paediatric/adult). e Overrepresented GO biological process terms (FDR < 0.05)

Fig. 6

An example of differential methylation at myeloid VDR peaks overlapping with MS risk genes. In cells of paediatric origin, DNA methylation was increased at IRF8 (left) and decreased at TNIP3 (right) relative to cells of adult origin. Both genes were also differentially expressed between cells of adult and paediatric origin (see Additional file 7). Black arrows denote differentially methylated regions. Red tracks: paediatric, blue tracks: adult

Fig. 7

A potential mechanism for the development of autoimmune disease risk dependent on early life vitamin D exposure. Decreased VDR binding site methylation in early life increases phenotypic plasticity and susceptibility to vitamin D exposure. Because tissue macrophages persist for months to years, phenotypic settings resulting from vitamin D exposure in early life may lead to a tolerogenic or autoimmune propensity in later life. Macrophage images adapted from [26]