Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-hydroxyvitamin D levels and autoimmune disease

Abstract

BackgroundVitamin D insufficiency has been implicated in autoimmunity. ChIP-seq experiments using immune cell lines have shown that vitamin D receptor (VDR) binding sites are enriched near regions of the genome associated with autoimmune diseases. We aimed to investigate VDR binding in primary CD4+ cells from healthy volunteers.MethodsWe extracted CD4+ cells from nine healthy volunteers. Each sample underwent VDR ChIP-seq. Our results were analyzed in relation to published ChIP-seq and RNA-seq data in the Genomic HyperBrowser. We used MEMEChIP for de novo motif discovery. 25-Hydroxyvitamin D levels were measured using liquid chromatography–tandem mass spectrometry and samples were divided into vitamin D sufficient (25(OH)D ≥75 nmol/L) and insufficient/deficient (25(OH)D <75 nmol/L) groups.ResultsWe found that the amount of VDR binding is correlated with the serum level of 25-hydroxyvitamin D (r = 0.92, P= 0.0005). In vivo VDR binding sites are enriched for autoimmune disease associated loci, especially when 25-hydroxyvitamin D levels (25(OH)D) were sufficient (25(OH)D ≥75: 3.13-fold, P<0.0001; 25(OH)D <75: 2.76-fold, P<0.0001; 25(OH)D ≥75 enrichment versus 25(OH)D <75 enrichment: P= 0.0002). VDR binding was also enriched near genes associated specifically with T-regulatory and T-helper cells in the 25(OH)D ≥75 group. MEME ChIP did not identify any VDR-like motifs underlying our VDR ChIP-seq peaks.ConclusionOur results show a direct correlation between in vivo 25-hydroxyvitamin D levels and the number of VDR binding sites, although our sample size is relatively small. Our study further implicates VDR binding as important in gene-environment interactions underlying the development of autoimmunity and provides a biological rationale for 25-hydroxyvitamin D sufficiency being based at 75 nmol/L. Our results also suggest that VDR binding in response to physiological levels of vitamin D occurs predominantly in a VDR motif-independent manner.

Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬

“…There is, moreover, a positive correlation between vitamin D levels and the number of VDR binding sites in the genome (32,34,35). For example, although in vitamin D-deficient individuals VDR binds to 601 sites in primary CD4+ T cells, this number increases to 4,518 (7.5-fold) in vitamin D-sufficient individuals (35).…”

“…There is, moreover, a positive correlation between vitamin D levels and the number of VDR binding sites in the genome (32,34,35). For example, although in vitamin D-deficient individuals VDR binds to 601 sites in primary CD4+ T cells, this number increases to 4,518 (7.5-fold) in vitamin D-sufficient individuals (35). Thus, a constant dietary vitamin D supplementation in rats, which we have previously shown to significantly increase levels of 25(OH)D, the major circulating form of vitamin D (23), may engage thousands of additional VDR binding sites, leading to marked changes in gene expression.…”

“…Patterns of VDR binding (20,35) and gene expression (39,51) in cell lines and healthy subjects have suggested an effect of vitamin D on metabolic and signaling pathways crucial for cell survival, growth, and proliferation. Our data confirm that these pathways are also affected in vivo in CD4+ T cells, mediating the protective effect of the vitamin D supplementation in experimental autoimmune disease.…”

Anti-inflammatory nutritional intervention in patients with relapsing-remitting and primary-progressive multiple sclerosis: A pilot study

“…Values higher than 75 nmol/L are considered to be adequate, because this is the minimal value at which vitamin D 3 is significantly bound to its receptor VDR, immunological properties become evident and the parathormone is reaching a plateau. [50][51][52] RDA equivalent doses of vitamin D were chosen for the treatment of MS patients, because they are the most frequent doses of administration in common praxis. So, the question arises why in the present study cholecalciferol supplementation failed to increase vitamin D 3 levels.…”

Emerging Vitamin D Receptor-Centered Patterns of Genetic Overlap across Some Autoimmune Diseases and Associated Cancers

“…Likely, a malfunction of VDR could affect the pathogenesis of RA and associated cancers expanding to many other ADs, Paraneoplastic Neurological Diseases(PND) and DM (Table 1) [1,13,14,16,[17][18][19][20][21][22][23][24]. In addition, VDR ChIP-seq in primary CD4+ cells relates serum 25-hydroxyvitamin D levels to autoimmune disease [25]. In closing, the patho-physiology of ADs (at least subgroup) may share their common underlying mechanisms of genetic regulatory network of VDR.…”