Gene-Regulatory Potential of 25-Hydroxyvitamin D3 and D2.

Human peripheral blood mononuclear cells (PBMCs) represent a highly responsive primary tissue that is composed of innate and adaptive immune cells. In this study, we compared modulation of the transcriptome of PBMCs by the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3), 25(OH)D2 and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Saturating concentrations of 1,25(OH)2D3, 25(OH)D3 and 25(OH)D2 resulted after 24 h stimulation in a comparable number and identity of target genes, but below 250 nM 25(OH)D3 and 25(OH)D2 were largely insufficient to affect the transcriptome. The average EC50 values of 206 common target genes were 322 nM for 25(OH)D3 and 295 nM for 25(OH)D2 being some 600-fold higher than 0.48 nM for 1,25(OH)2D3. The type of target gene, such as primary/secondary, direct/indirect or up-/down-regulated, had no significant effect on vitamin D metabolite sensitivity, but individual genes could be classified into high, mid and lower responders. Since the 1α-hydroxylase CYP27B1 is very low expressed in PBMCs and early (4 and 8 h) transcriptome responses to 25(OH)D3 and 25(OH)D2 were as prominent as to 1,25(OH)2D3, both vitamin D metabolites may directly control gene expression. In conclusion, at supra-physiological concentrations 25(OH)D3 and 25(OH)D2 are equally potent in modulating the transcriptome of PBMCs possibly by directly activating the vitamin D receptor.

Time-Resolved Gene Expression Analysis Monitors the Regulation of Inflammatory Mediators and Attenuation of Adaptive Immune Response by Vitamin D.

Peripheral blood mononuclear cells (PBMCs) belong to the innate and adaptive immune system and are highly sensitive and responsive to changes in their systemic environment. In this study, we focused on the time course of transcriptional changes in freshly isolated human PBMCs 4, 8, 24 and 48 h after onset of stimulation with the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Taking all four time points together, 662 target genes were identified and segregated either by time of differential gene expression into 179 primary and 483 secondary targets or by driver of expression change into 293 direct and 369 indirect targets. The latter classification revealed that more than 50% of target genes were primarily driven by the cells' response to ex vivo exposure than by the nuclear hormone and largely explained its down-regulatory effect. Functional analysis indicated vitamin D's role in the suppression of the inflammatory and adaptive immune response by down-regulating ten major histocompatibility complex class II genes, five alarmins of the S100 calcium binding protein A family and by affecting six chemokines of the C-X-C motif ligand family. Taken together, studying time-resolved responses allows to better contextualize the effects of vitamin D on the immune system.

Vitamin D Treatment Sequence Is Critical for Transcriptome Modulation of Immune Challenged Primary Human Cells.

Microbe-associated molecular patterns, such as lipopolysaccharide (LPS) and ß-glucan (BG), are surrogates of immune challenges like bacterial and fungal infections, respectively. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), supports the immune system in its fight against infections. This study investigated significant and prominent changes of the transcriptome of human peripheral blood mononuclear cells that immediately after isolation are exposed to 1,25(OH)2D3-modulated immune challenges over a time frame of 24-48 h. In this in vitro study design, most LPS and BG responsive genes are downregulated and their counts are drastically reduced when cells are treated 24 h after, 24 h before or in parallel with 1,25(OH)2D3. Interestingly, only a 1,25(OH)2D3 pre-treatment of the LPS challenge results in a majority of upregulated genes. Based on transcriptome-wide data both immune challenges display characteristic differences in responsive genes and their associated pathways, to which the actions of 1,25(OH)2D3 often oppose. The joined BG/1,25(OH)2D3 response is less sensitive to treatment sequence than that of LPS/1,25(OH)2D3. In conclusion, the functional consequences of immune challenges are significantly modulated by 1,25(OH)2D3 but largely depend on treatment sequence. This may suggest that a sufficient vitamin D status before an infection is more important than vitamin D supplementation afterwards.

Transcriptome-Wide Profile of 25-Hydroxyvitamin D3 in Primary Immune Cells from Human Peripheral Blood.

Vitamin D3 is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25(OH)D3), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and <0.05 as a false discovery rate, in this ex vivo approach the maximal physiological concentration of 25(OH)D3 (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D3 regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH)2D3. The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D3. In conclusion, 100- and 1000-fold higher 25(OH)D3 concentrations than the reference 10 nM 1,25(OH)2D3 are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH)2D3.

Common and personal target genes of the micronutrient vitamin D in primary immune cells from human peripheral blood.

Vitamin D is essential for the function of the immune system. In this study, we treated peripheral blood mononuclear cells (PBMCs) of healthy adults with the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) using two different approaches: single repeats with PBMCs obtained from a cohort of 12 individuals and personalized analysis based on triplicates of five study participants. This identified 877 (cohort approach) and 3951 (personalized approach) genes that significantly (p < 0.05) changed their expression 24 h after 1,25(OH)2D3 stimulation. From these, 333 and 1232 were classified as supertargets, a third of which were identified as novel. Individuals differed largely in their vitamin D response not only by the magnitude of expression change but also by their personal selection of (super)target genes. Functional analysis of the target genes suggested the overarching role of vitamin D in the regulation of metabolism, proliferation and differentiation, but in particular in the control of functions mediated by the innate and adaptive immune system, such as responses to infectious diseases and chronic inflammatory disorders. In conclusion, immune cells are an important target of vitamin D and common genes may serve as biomarkers for personal responses to the micronutrient.

Skin colour and vitamin D: An update.

Homo sapiens evolved in East Africa and had dark skin, hair, and eyes, in order to protect against deleterious consequences of intensive UV radiation at equatorial latitudes. Intensive skin pigmentation was thought to bear the risk of inefficient vitamin D3 synthesis in the skin. This initiated the hypothesis that within the past 75 000 years, in which humans migrated to higher latitudes in Asia and Europe, the need for vitamin D3 synthesis served as an evolutionary driver for skin lightening. In this review, we summarize the recent archeogenomic reconstruction of population admixture in Europe and demonstrate that skin lightening happened as late as 5000 years ago through immigration of lighter pigmented populations from western Anatolia and the Russian steppe but not primarily via evolutionary pressure for vitamin D3 synthesis. We show that variations in genes encoding for proteins being responsible for the transport, metabolism and signalling of vitamin D provide alternative mechanisms of adaptation to a life in northern latitudes without suffering from consequences of vitamin D deficiency. This includes hypotheses explaining differences in the vitamin D status and response index of European populations.

Key Vitamin D Target Genes with Functions in the Immune System.

The biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), modulates innate and adaptive immunity via genes regulated by the transcription factor vitamin D receptor (VDR). In order to identify the key vitamin D target genes involved in these processes, transcriptome-wide datasets were compared, which were obtained from a human monocytic cell line (THP-1) and peripheral blood mononuclear cells (PBMCs) treated in vitro by 1,25(OH)2D3, filtered using different approaches, as well as from PBMCs of individuals supplemented with a vitamin D3 bolus. The led to the genes ACVRL1, CAMP, CD14, CD93, CEBPB, FN1, MAPK13, NINJ1, LILRB4, LRRC25, SEMA6B, SRGN, THBD, THEMIS2 and TREM1. Public epigenome- and transcriptome-wide data from THP-1 cells were used to characterize these genes based on the level of their VDR-driven enhancers as well as the level of the dynamics of their mRNA production. Both types of datasets allowed the categorization of the vitamin D target genes into three groups according to their role in (i) acute response to infection, (ii) infection in general and (iii) autoimmunity. In conclusion, 15 genes were identified as major mediators of the action of vitamin D in innate and adaptive immunity and their individual functions are explained based on different gene regulatory scenarios.

Genome-wide effects of chromatin on vitamin D signaling.

Molecular endocrinology of vitamin D is based on the activation of the transcription factor vitamin D receptor (VDR) by the vitamin D metabolite 1α,25-dihydroxyvitamin D3. This nuclear vitamin D-sensing process causes epigenome-wide effects, such as changes in chromatin accessibility as well as in the contact of VDR and its supporting pioneer factors with thousands of genomic binding sites, referred to as vitamin D response elements. VDR binding enhancer regions loop to transcription start sites of hundreds of vitamin D target genes resulting in changes of their expression. Thus, vitamin D signaling is based on epigenome- and transcriptome-wide shifts in VDR-expressing tissues. Monocytes are the most responsive cell type of the immune system and serve as a paradigm for uncovering the chromatin model of vitamin D signaling. In this review, an alternative approach for selecting vitamin D target genes is presented, which are most relevant for understanding the impact of vitamin D endocrinology on innate immunity. Different scenarios of the regulation of primary upregulated vitamin D target genes are presented, in which vitamin D-driven super-enhancers comprise a cluster of persistent (constant) and/or inducible (transient) VDR-binding sites. In conclusion, the spatio-temporal VDR binding in the context of chromatin is most critical for the regulation of vitamin D target genes.

Vitamin D and evolution: Pharmacologic implications.

Vitamin D3 is produced non-enzymatically when the cholesterol precursor 7-dehydrocholesterol is exposed to UV-B, i.e., evolutionary the first function of the molecule was that of an UV-B radiation scavenging end product. Vitamin D endocrinology started when some 550 million years ago first species developed a vitamin D receptor (VDR) that binds with high affinity the vitamin D metabolite 1α,25-dihydroxyvitamin D3. VDR evolved from a subfamily of nuclear receptors sensing the levels of cholesterol derivatives, such as bile acids, and controlling metabolic genes supporting cellular processes, such as innate and adaptive immunity. During vertebrate evolution, the skeletal and adaptive immune system showed in part interesting synchronous development although adaptive immunity is evolutionary older. There are bidirectional osteoimmune interactions between the immune system and bone metabolism, the regulation of both is under control of vitamin D. This diversity of physiological functions explains the pleiotropy of vitamin D signaling and opens the potential for various pharmacological applications of vitamin D as well as of its natural and synthetic derivatives. The overall impact of vitamin D on human health is demonstrated by the fact that the need for its efficient synthesis served in European hunter and gatherers as an evolutionary driver for increased 7-dehydrocholesterol levels, while light skin was established far later via populations from Anatolia and the northern Caucasus entering Europe 9000 and 5000 years ago, respectively. The later population settled preferentially in northern Europe and we hypothesize that that the introduction of high vitamin D responsiveness was an essential trait for surviving dark winters without suffering from the detrimental consequences of vitamin D deficiency.