Neuromol Med (2014) 16:265–279 DOI 10.1007/s12017-014-8301-2

REVIEW PAPER

Genomic Binding Sites and Biological Effects of the Vitamin D: VDR Complex in Multiple Sclerosis Bernadette Kalman • Erzsebet Toldy

Received: 11 January 2014 / Accepted: 29 March 2014 / Published online: 8 April 2014  Springer Science+Business Media New York 2014

Abstract Environmental factors greatly contribute to the development of complex trait disorders. With the rapid developments in the fields of biotechnology and informatics, the investigations of molecular interactions between host and environmental factors became very detailed and comprehensive. The effects of ultraviolet irradiation, vitamin D synthesis, and receptor binding, and then the involvements of the ligand–receptor complexes in the regulation of cell function received much attention in the last few years and paralleled the accumulation of information concerning genetic determinants of disease susceptibility, transcriptional regulation, cell cycle, mitochondrial biochemistry, and many other elements of cell biology. The importance of vitamin D in contributing to immune regulation, autoimmunity, and susceptibility to multiple sclerosis (MS) is now also recognized, and there are ongoing treatment trials with vitamin D to define whether it is capable of modifying susceptibility to or the course of the disease. This survey aims to capture that part of vitamin D research that helps to better understand the interactions of this molecule and its receptor with the human genome, and the downstream effects of these interactions relevant to immune homeostasis and MS. This relatively narrow scope reveals a very complex molecular network underlying inflammatory demyelination and allows us to hope that a better understanding of the roles of vitamin D and other environmental factors will once make

B. Kalman (&)  E. Toldy Faculty of Health Sciences, University of Pecs, Center for Research and Education, Markusovszky University Teaching Hospital, Markusovszky Street 5, 9700 Szombathely, Hungary e-mail: [email protected]; [email protected]

the risk of MS modifiable or, to some degrees, the disease preventable. Keywords Vitamin D receptor  Genomic binding sites  Transcriptional regulation  Autoimmunity  Multiple sclerosis

Introduction Vitamin D is a lipid-soluble vitamin with multiple biological functions involved in calcium and phosphorus homeostasis, bone mineralization, immune regulation, and many cellular processes regulating cancer development, autoimmunity, and longevity. Intake of vitamin D2 and D3 with food and production of previtamin D3 from 7-dehydrocholesterol upon ultraviolet (UV) irradiation in the skin represent its two main sources (Fig. 1). The biologically active form of the vitamin is generated in two subsequent steps. First, vitamin D2 and previtamin D3 are hydroxylated in the liver to produce the intermediate and biologically inactive 25-hydroxyvitamin D or 25(OH)D. In the second step, 25(OH)D is hydroxylated further by the enzyme called 25(OH)D3-1a-hydroxylase to generate the active form, 1a,25-dihydroxy-vitamin D or 1a,25(OH)2D3 in the kidney. The 25(OH)D3-1a-hydroxylase is also known as cytochrome p450 27B1 or CYP27B1. This enzyme had been thought to be only expressed in the kidney under the control of parathyroid hormone and other calcium-regulatory molecules. However, recent data show that CYP27B1 is also expressed in non-renal tissues and controlled by a variety of signals (Dimitrov et al. 2013). The active form of vitamin D, 1a,25(OH)2D3, is also called calcitriol. In vivo, 1a,25(OH)2D3 binds to the vitamin D receptor (VDR) that forms dimers with the retinoid X receptors (RXR). VDR is

123

266

Neuromol Med (2014) 16:265–279

Fig. 1 Production of the biologically active form of vitamin D and its main actions. The figure shows sources, synthesis, and main actions of the biologically active form of vitamin D3

a member of the nuclear receptor superfamily characterized by activation upon exposure to pico to nanomolar concentrations of their ligands. The 1a,25(OH)2D3–VDR complex exerts its effect by binding to one of the Vitamin D response elements (VDRE) in the genome and is involved in gene expression regulation (Fig. 2). The preferentially recognized VDRE in the genome is the classical motif of DR3, a direct repeat of two hexameric core sequences spaced by 3 nucleotides. However, this motif is present at only a proportion of VDR-binding sites as determined by genome-wide VDR-binding analyses in various cell types. Other, less frequently occupied VDRE motifs and non-VDRE motifs are also utilized (Heikkinen et al. 2011; Dimitrov et al. 2013). The DNA-bound VDR complex recruits coactivators and corepressors involved in chromatin remodeling and transcription regulation (Fig. 2) (Dimitrov et al. 2013). In the classical model, the VDR binding to VDREs leads to transcriptional activation, but mechanisms of transcriptional repression are more heterogeneous and often involve interactions of the VDR with other transcription factors. The genomic distribution of VDR binding is cell- and tissue-specific as demonstrated in several studies (Ramagopalan et al. 2010; Disanto et al. 2012a; Handel et al. 2013; Heikkinen et al. 2011; Dimitrov et al. 2013). In the immune system, the gene expression regulation by vitamin D generally contributes to enhanced defense mechanisms

123

upon microbial challenges in the innate immune system, and to tolerance induction ensuring protection from autoimmunity and allergies in the adaptive immune system (Verstuyf et al. 2010; Zittermann et al. 2009). In addition to binding to VDREs in the nucleus, VDR can also control genomic targets without forming a complex with its vitamin D ligand, when it associates via corepressor proteins with histone deacetylases. Ligand binding causes conformational changes and dissociation from the corepressor. Thus, VDR may have dual functioning: it can modulate chromatin opening when associates with corepressors and histone proteins, or may regulate gene transcription when forms complex with its ligand and other nuclear factors at or in the proximity of transcription start sites (TSS) of target genes (Fig. 2) (Heikkinen et al. 2011). This paper is focused on the main interactions of vitamin D/VDR with the genome and highlights some of the well-characterized VDR-mediated mechanisms of gene expression regulation in various conditions, with emphasis on multiple sclerosis (MS).

Multiple Sclerosis Multiple sclerosis is characterized by inflammation, demyelination, and neurodegeneration in the central nervous system (CNS). The etiology remains unknown, but

Neuromol Med (2014) 16:265–279

267

Fig. 2 Some of the main actions of the VDR and VDR– ligand complex. This figure depicts the main molecular interactions that involve the vitamin D/VDR complex or the VDR alone in various biological processes

several lines of evidence support the involvements of immune dysregulation and autoimmunity in disease pathogenesis. Epidemiological studies revealed familial recurrence of the disease in about 15 % of patients and underscored that the inheritance is not compatible with mendelian or mitochondrial transmission patterns (Compston 1999). MS is a complex trait disease defined by interactions among several genes and environmental factors. The polygenic determination of susceptibility is supported by the results of epidemiologic, case–control candidate gene, linkage, and genome-wide association studies (GWAS) (Baranzini 2011). The first GWAS published in 2007 underscored the importance of three MS risk variants within the human leukocyte antigen (HLA) locus and the interleukin 7 receptor (IL7R) and IL2RA genes (Hafler et al. 2007). Subsequently, seven GWAS studies and one meta-analysis were reported, which implicated 57 non-HLA MS risk loci by 2013 (The International Multiple Sclerosis Consortium and The Wellcome Trust Case Control Consortium 2, 2011, The International Multiple Sclerosis Genetics Consortium 2013a). The latest GWAS involving more than 80,000 subjects, added another 48 single-nucleotide polymorphism (SNP) susceptibility variants to the previous list, and extended the number of nonHLA MS risk variants to 110 at 101 loci (The International Multiple Sclerosis Consortium 2013b). Among all candidate genes, the major histocompatibility complex (MHC or

HLA) locus contributes the highest risk to susceptibility and may define 40–50 % of MS heredity (The International Multiple Sclerosis Genetics Consortium 2013a). Pathway analyses of risk genes and their protein products reveal that most of the identified candidates are involved in cellular immune regulation, apoptosis, and energy metabolism as well as in neuronal processes (Baranzini et al. 2009; The International Multiple Sclerosis Genetics Consortium 2013a). Altogether, the observations from hypothesis-driven and hypothesis-free studies have provided unequivocal support to the autoimmune etiology of MS and identified the specific SNP variants that modify the risk for the disease. While each of these SNPs exerts only a small effect, their combined effect defines the complex trait underlying MS. However, genetic heredity can only explain part of MS susceptibility, most eloquently demonstrated in studies on monozygotic twins. In genetically identical monozygotic twins, the observed concordance rates are only about 25 % (34 % in female pairs) (Willer et al. 2003), suggesting a marked involvement of non-genetic determinants influencing disease risk. Environmental factors may significantly influence gene expression regulation and cell function via chromatin modification (histone acetylation, methylation, ubiquitination, sumoylation, etc.…), CpG methylation, gene–gene and protein–protein interactions, or micro-RNA-mediated processes. Environmental factors

123

268

also may act as ligands for nuclear factors involved in gene transcription regulation. Through these mechanisms, environmental factors significantly contribute to defining clinical manifestation of complex diseases. In MS, viruses (Ascherio and Munger 2010; Virtanen and Jacobson 2012), tobacco smoking (Simon et al. 2010a, b; Salzer and Sundstro¨m 2013; Ramagopalan et al. 2013), and vitamin D (Ascherio et al. 2010; Simon et al. 2012; Smolders et al. 2009, 2011, 2013, 2014) have been most consistently identified as environmental determinants of susceptibility. The contribution of vitamin D to MS is supported by epidemiological studies (Ascherio et al. 2010; Simon et al. 2010a, b), measurements of vitamin D and vitamin D-binding protein (DBP) serum levels (Simon et al. 2010a, b) (although not showing a clear association between DBP and MS by Smolders et al. 2014), the demonstration of the effects of vitamin D on the activity of immune cells (Simon et al. 2010a, b; Smolders and Damoiseaux 2011; Knippenberg et al. 2011), and the detection of rare MS-associated variants in the CYP27B1 gene involved in the metabolism of vitamin D (Ramagopalan et al. 2011). A study on MS and control brain tissues also revealed the expression differences of VDR and vitamin D-metabolizing enzymes (24-OHase or CYP24A1 and 25(OH)D1a-hydroxylase or CYP27B1) in normal appearing white matter (NAWM), chronic active and inactive lesions of MS, and in NAWM of patients and white matter of controls (Smolders et al. 2013). The authors interpreted that the increased VDR expression in NAWM, and the proinflammatory cytokine-driven enhanced expression of VDR and CYP27B1 in chronic active lesions, may reflect an increased sensitivity to vitamin D in NAWM and an anti-inflammatory effect of vitamin D in active MS lesions (Smolders et al. 2013). In addition, several treatment trials have shown some effect of vitamin D administration in various stages and forms of MS (Do¨rr et al. 2012; James et al. 2013), while some trials are still in progress (Do¨rr et al. 2013; Smolders et al. 2011). However, the exact clinical benefits of vitamin D in modulating susceptibility to inflammatory demyelination or its course remain to be determined (Do¨rr et al. 2013). Altogether, the epidemiological and clinical observations in MS raised the question as to what molecular mechanisms are being utilized by vitamin D to modulate the immune system and susceptibility to the disease. The answers to this question came from several lines of investigations including the studies of VDR binding in the genome under various conditions (e.g., in unstimulated and vitamin D-stimulated cell lines), in relation to the location of SNPs implicated in defining disease susceptibility, and in relation to chromatin status, transcriptional start sites (TSS), and expression regulation of target genes. Immune regulatory effects of VDR in immune cells are reviewed in

123

Neuromol Med (2014) 16:265–279

details by Mora´n-Auth et al. (2013). Here, we focus on those vitamin D/VDR-regulated immune functions that are most relevant to MS and its animal model, experimental autoimmune encephalomyelitis (EAE). EAE is an inflammatory demyelinating disease of the central nervous system (CNS), most frequently induced in mice and rats for studying certain aspects of MS. As a model, it has been strongly criticized for involving a completely different trigger of disease pathogenesis than MS, and therefore, causing a biased concept concerning the etiology of the human disease. In addition, there are major differences in phenotypic as well as functional features of the immune system in humans and rodents. Nevertheless, EAE is still probably the best model that allows us studying the mechanisms of immune regulation, inflammation-induced demyelination, and neurodegeneration as well as the effects of disease-modifying therapeutic interventions relevant to MS. Active EAE is typically induced by subcutaneous injection of a large dose of a myelin-related protein or antigenic peptide combined with complete Freund’s adjuvant to overcome both central and peripheral tolerance. The disease is primarily mediated by myelin-related protein or peptide-specific autoimmune T cells that migrate from the periphery to the CNS and cause inflammation and demyelination. While several elements of the adaptive immune system drive the development of the disease, the innate immune system also plays important roles in defining pathogenesis. Since VDR is expressed by a variety of immune cells including monocytes, macrophages, dendritic cells, NK, and T and B cells, the regulatory roles of 1a,25(OH)2D3 have been extensively investigated in both the innate and adaptive immune components of EAE (see below). These investigations into the model system were further supported by the observations that 1a,25(OH)2D3 not only modulates the course of MS, but also influences the clinical outcome of EAE (Cantorna et al. 1996; Nashold et al. 2000; Chang et al. 2010; Adzemovic et al. 2013; Grishkan et al. 2013).

VDR: Genome Interactions Several MS investigators aimed elucidating the roles of VDR in gene expression regulation. As a first approach, they used the method of chromosome immune precipitation and massively parallel sequencing (ChIP-seq) to identify binding sites of the VDR in the genome and then applied the Genome HyperBrowser program to determine the overlap between GWAS-identified MS-relevant SNPs and VDR genomic bindings (Ramagopalan et al. 2010; Disanto et al. 2012a; Handel et al. 2013). These studies were carried out in B-cell lines and CD4 T lymphocytes (Ramagopalan et al. 2010; Disanto et al. 2012a; Handel et al.

Neuromol Med (2014) 16:265–279

2013). A similar genome-wide analysis of VDR chromatin occupancy before and after vitamin D treatment in a monocytic leukemia cell line was published by Heikkinen et al. (2011). Testing various cell types (B- and monocytic cell lines, healthy CD4 T lymphocytes) was important because of the cell- and tissue-specific characteristics of chromatin landscapes underlying distinct transcriptional and functional profiles (Ernst et al. 2011). In the first study using the method of ChIP-seq in LCL (B-lymphoblastic cell line), Ramagopalan et al. (2010) detected 2776 VDR bindings genome-wide following calcitriol stimulation, while only 623 genomic regions were occupied by VDR in the unstimulated state. In unstimulated state, half of the binding sites appeared to be localized to promoter regions, while the rest of the binding sites were shared among intergenic, intronic, upstream, downstream, untranslated, and coding DNA sequence regions. Upon calcitriol stimulation, the increase in VDR-binding sites was accompanied by a shift toward intronic and intergenic regions. Investigating the sequence characteristics of VDR binding identified the DR3 motif as the most significant VDRE enrichment motif. Microarray analyses of transcripts from the calcitriol-stimulated LCLs revealed 226 significantly upregulated and 3 downregulated genes that were found enriched for immune function in pathway analyses. Twenty-three % of vitamin D-responsive genes had VDR intervals near to transcription start sites, and 96 % of these intervals had a DR3 motif. VDR-binding sites were significantly enriched near to MS-associated genetic loci. However, other common anthropological and disease trait loci (e.g., height, hair color, skin sensitivity to sun, type 1 diabetes, Crohn’s disease, rheumatoid arthritis, lupus erythematosis, and colorectal cancer) were also enriched for VDR binding, but each trait showed a distinct pattern of VDR-binding profile. Since subsequent GWAS studies revealed additional MS susceptibility variants, Disanto et al. (2012a) performed a new Chip-seq analysis to elucidate the relationship between VDR binding and chromatin states in LCL, and analyzed their relation to the extended susceptibility loci of MS. VDR-binding regions significantly overlapped with active promoter (AP) and strong enhancer (SE) regions in LCL more often than expected by chance, as close to 80 % of VDR-binding regions had either AP or SE elements (Disanto et al. 2012a). Interestingly, these colocalizations of VDR-binding regions and APs or SEs were significantly greater in LCLs than in non-immune cells and were enriched within MS susceptibility genetic regions and other disease regions revealed by various GWAS. More than 60 % of MS susceptibility regions appeared to be bound by VDR. The VDR-AP and VDR-SE elements were not only found to be located in the same genomic regions, but they also tended to overlap at nucleotide level. This observation

269

suggests that the VDRs utilize the enhancer and promoter elements and, through that, directly influence gene expression regulation. The finding that MS-associated genomic regions are characterized by intense transcriptional activity in B-lymphoblastoid cell lines (Disanto et al. 2012b) supports the assumption that disease-related genetic variants should be located within transcriptionally active regions in the disease-relevant (in this case B) cell type(s). Experimental evidence suggests that in B-cell lineages, vitamin D modulates cell proliferation, apoptosis, plasma cell differentiation, and immunoglobulin synthesis (Chen et al. 2007). These studies also highlight the interplays between chromatin states and VDR bindings in MS susceptibility loci, and provide a biological mechanism for explaining as to how vitamin D may contribute to defining immunopathology of the disease. Subsequently, Handel et al. (2013) reported VDR ChIPseq and RNA-seq analyses in primary CD4 T cells of nine healthy volunteers. Vitamin D serum levels were also determined, and the samples were divided into vitamin D-sufficient (25(OH)D = /[75 nmol/L) and vitamin D-deficient (25(OH)D \ 75 nmol/L) groups. There were several noteworthy findings, they are as follows: (1) the amount of VDR binding correlated with the serum 25(OH)D levels; (2) in vivo VDR-binding sites were enriched in regions of genetic susceptibility determinants of autoimmune diseases, particularly in 25(OH)D-sufficient individuals; and (3) VDR binding was also enriched near genes associated with T-regulatory and T helper cell function in the 25(OH)D-sufficient group. This genomewide study in a small group of healthy individuals emphasizes the correlation between in vivo 25(OH)D levels and the number of VDR-binding sites, while further supports that environmental factors, such as vitamin D intake, may contribute to autoimmunity by impacting on VDR binding and gene expression regulation, particularly in cells of adaptive immunity. Curiously, VDR binding in this study occurred in a VDR motif-independent manner in response to physiological levels of vitamin D (Handel et al. 2013). The authors postulate that in vivo VDR binding may be modulated by protein–protein interactions with cofactors such as SP1 (specificity protein 1), NR4A1 (nuclear receptor subfamily 4 group A member 1), and c-MYC or CTCF (CCCTC-binding factor). The genomic landscape may also significantly influence the in vivo VDR binding suggested by the overlap with sites sensitive to DNase I (Bernstein et al. 2012). However, interactions with other factors (e.g., serum parathyroid hormone and calcium levels) might have influenced the above findings. In addition to the studies by the MS collaborative group in the United Kingdom (Ramagopalan et al. 2010; Disanto et al. 2012a; Handel et al. 2013), Heikkinen et al. (2011) independently published similar observations. These

123

270

authors also performed VDR-binding analyses genomewide using the ChIP-seq approach complemented with expression profiling by microarray analyses in a monocytic leukemia line (THP-1) maintained in the presence or absence of 1a,25(OH)2D3 treatment. Comparable to data by Ramagopalan et al. (2010), this study identified 2,340 VDR-binding sites, which included 520 unique sites in unstimulated, 1,171 unique sites in stimulated condition, and 649 common sites. The DR3 motif occupancy by VDR was strongly associated with the ligand responsiveness. The proportion of most decreasing VDR sites with DR3 motif was 20 %, and of the most increasing VDR sites with DR3 motif was 90 % after 1a,25(OH)2D3 treatment, indicating a shift toward the DR3 VDRE occupancy by VDR upon ligand exposure. Gene ontology studies detected 638 vitamin D-targeted genes enriched in immune and signaling functions. Almost three-fourths of the 408 upregulated genes had VDR binding within 400 kb of their TSSs, while this was true only for less than half of the downregulated genes. VDR binding was observed in various patterns ranging from single VDR binding near the TSS of a target gene to complex VDR-binding clusters and target genes. Although these studies were carried out in a different cell line (monocytic leukemia line) than the previous ones, many aspects of the observations are in consensus with those of Ramagopalan et al. (2010), Disanto et al. (2012a), and Handel et al. (2013) obtained from a B-lymphoblastic cell line and normal CD4 T lymphocytes. Similarly, Satoh and Tabunoki (2013) also studied genome-wide VDR target genes by identifying VDRbinding sites with the ChIP-seq method in calcitriol-treated human cells of B cell and monocyte origins. The authors found 2997 VDR-binding sites distributed on proteincoding genes, mostly located at VDRE DR3 sequences in promoter and intronic regions. However, the transcriptome data showed calcitriol-induced upregulation of only a proportion of VDR target genes. The 1541 calcitriolresponsive VDR target genes clustered in immune regulatory functional groups including leukocyte transendothelial migration, Fcc receptor-mediated phagocytosis, and transcriptional regulation by VDR. Highly relevant to MS, a strongly conserved VDRE sequence had been identified within the promoter of the MS-associated HLA DRB1*1501 and involved in the expression regulation of this MHC class II molecule on immune cells (Ramagopalan et al. 2009; Handunnetthi et al. 2010). Specific recruitment of VDR to the VDRE in the HLA-DRB1*15 promoter was detected by electrophoretic mobility shift assay and confirmed by ChIP in lymphoblastoid cells. Further, B cells transiently transfected with the HLA-DRB1*15 gene promoter demonstrated increased expression of the gene upon stimulation with 1a,25(OH)2D3, which was lost when the VDRE sequence

123

Neuromol Med (2014) 16:265–279

was deleted or when the homologous ‘‘VDRE’’ sequences of non-MS-associated HLA-DRB1 haplotypes were used. Exposure to 1a,25(OH)2D3 induced an increased cell surface expression of HLA-DRB1 only in HLA-DRB1*15positive lymphoblastoid cells. These studies provide strong evidence to support the specific interaction between vitamin D, an environmental factor, and the MS-associated MHC DRB1*15 gene allele (Ramagopalan et al. 2009; Handunnetthi et al. 2010). In the meantime, other studies also revealed MS-relevant genetic variants in the VDR gene. However, these variants did not seem to influence the HLA DRB1*15 promoter VDRE and VDR interactions, rather appeared to modulate vitamin D metabolism and gene expression regulation (Tajouri et al. 2005; Smolders et al. 2009). Further, VDR SNP variants did not show association with MS (Simon et al. 2010a, b; Garcia-Martin et al. 2013). Altogether, these studies have elucidated several aspects of the vitamin D/VDR interactions with the genome and revealed its potential involvement in immune dysregulation, autoimmunity, and MS pathogenesis.

Complex Mechanisms of Gene Expression Regulation by VDR in Various Conditions As mentioned above, the chromatin landscape influences the VDR-induced gene expression regulation. Seuter et al. (2013) investigated how chromatin acetylation at TSS and at VDR-binding regions relates to the effects of histone deacetylase inhibitors and 1a,25(OH)2D3-mediated gene expression. The effects of trichostatin A (TsA), an inhibitor of histone deacetylases, and of 1a,25(OH)2D3/VDR were studied on a number of reference genes and the thrombomodulin gene locus. The latter has five VDR-binding sites and multiple histone acetylation and open chromatin regions. Within the entire transcriptome, 18.4 % of the expressed genes were found to be up- or downregulated by a 90-min exposure to TsA. When compared to 1a,25(OH)2D3, TsA stimulated much more many genes during, and dominated, the outcome of the combined exposures. While 200 TsA-regulated genes were also modulated by 1a,25(OH)2D3, 1,000 genes responded only when exposed to both agents. The study also showed that the degree of histone acetylation at TSSs and VDR-binding regions determined the effect of TsA on gene transcription and its co-regulatory effect with 1a,25(OH)2D3. Not surprisingly, the 1a,25(OH)2D3/VDR-mediated regulation of target genes appeared to be much more specific than gene expression regulation by the histone deacetylase inhibitor TsA. Besides showing an important link between histone modification and VDR-mediated gene expression regulation, these data suggest a potential utility of dual therapies

Neuromol Med (2014) 16:265–279

including chromatin modifiers and nuclear receptor ligands in conditions associated with complex patterns of altered gene expression regulation (Seuter et al. 2013). Not only determines the state of chromatin the binding of VDR to accessible genomic sites (e.g., with acetylated histone), but vice versa, the binding of the VDR–DNA and activation of transcription also alter the chromatin environment reflected by increases and decreases in histone modifications (Meyer et al. 2013). The 1a,25(OH)2D3induced histone profile was recently studied to discover VDR action sites at individual genes (Meyer et al. 2013). This study revealed that VDR binds selectively to an enhancer 10 kb upstream of the TSS of the matrix metalloproteinase (MMP)-13 gene Mmp13, which is accompanied by an enhancer-selective increase in histone 3 lysine 9 acetylation (H3K9Ac) in mesenchymal stem cells treated with 1a,25(OH)2D3. Since the Mmp13 expression upregulation was noted in osteoblasts and mesenchymal stem cells as well as in their differentiating lineages, it was postulated that either a multifunctional enhancer or several enhancers bind distinct transcription factors in various cells and various conditions. The authors reported that several enhancer regions are present within the Mmp13 gene and characterized by distinct histone profiles. One of them has a 1a,25(OH)2D3-induced H3K9Ac binding. Thus, the 1a,25(OH)2D3-induced histone modification (H3K8Ac) points to an enhancer on which VDR specifically acts 10 Kb upstream from the TSS of Mmp13 (Meyer et al. 2013). The variability of mechanisms involved in transcription regulation by the VDR is well reflected by the survey of Dimitrov et al. (2013) in the context of calcium homeostasis, cancer prevention, and immune regulation. The VDR ligand 1a,25(OH)2D3 inhibits the expression of CYP27B1 in the kidney and parathormone in the parathyroid gland, which is the main inducer of renal CYP27B1. The mechanisms of transcriptional repression are similar in both cases and involve the association of VDR with a transcription factor (TF) called VDR-interacting repressor (VDIR). VDIR acts as a transactivator of the CYP27B2 gene in the absence of the VDR by binding to certain motifs in the promoter. Exposure to 1a,25(OH)2D3 results in the repression of the CYP27B1 gene by the complex of the interacting VDR and VDIR proteins (Murayama et al. 2004). A similar mechanism leads to the VDR-induced repression of the parathormone transcription by the association between VDR and VDIR in the gene promoter (Kim et al. 2007). The 1a,25(OH)2D3-induced interaction of VDR and VDIR also prevents the associations with coactivators and the recruitment of histone deacetylases along with other molecules involved in transcriptional repression (Dimitrov et al. 2013; Murayama et al. 2004; Kim et al. 2007). Calcium homeostasis is thus regulated by

271

this negative feedback and mediated by complex molecular interactions among 1a,25(OH)2D3, VDR, VDIR, histone deacetylases, and numerous transcriptional enhancers and repressors. In addition, there are AP1 sites within the VDRE in the promoter of osteocalcin gene, which may directly enhance osteocalcin transcription induced by the 1a,25(OH)2D3–VDR binding (Aslam et al. 1999). Another regulatory pathway modulated by VDR and VDR-ligand-induced signaling involves the FoxO protein family that controls cell cycle and differentiation in health and disease (Schmidt et al. 2002; Brennan et al. 1987; Lin et al. 2003; An et al. 2010). FoxO tumor suppressor proteins have posttranslational regulation through phosphorylation and acetylation. FoxO proteins are inhibited by the PI3 kinase through its interactions with Akt causing Aktdependent phosphorylation/nuclear export of FoxO proteins. The actions of the PI3 kinase can be reversed by phosphatases (Barreyro et al. 2007). In PTEN-deficient cancer, PI3 is unantagonized, causing a cytoplasmic retention of the FoxO proteins in an inactive state. The activity of FoxO proteins is also controlled by acetylation that can be reversed by sirtuin 1 (Sirt1), a deacetylase (Daitoku et al. 2004). Acetylation diminishes the target DNA binding of FoxO and increases its phosphorylation as well as inactivation. An et al. (2010) showed that VDR, independent of its ligand, directly associates with FoxO proteins, and regulators, Sirt1, and protein phosphatase 1. The interactions of VDR with FoxO proteins and cofactors significantly influence the functions of FoxO proteins in cell cycle regulation. In experimental conditions, 1a,25(OH)2D3 induces FoxO protein deacetylation and dephosphorylation that result in the active form and an increased binding of FoxO proteins to the promoters of their target genes (An et al. 2010). ChIP-seq studies also showed the presence of VDR and Sirt1 on FoxO target genes. The suppression of FoxO or Sirt1 gene transcription reduced the 1a,25(OH)2D3-dependent regulation of VDR/ FoxO target genes (An et al. 2010; Dimitrov et al. 2013). Cell cycle arrest by 1a,25(OH)2D3 did not occur in FoxO3a-deficient cells, suggesting that members of this protein family are important mediators of the vitamin D anti-proliferative effect (An et al. 2010, 1987; Dimitrov et al. 2013). These findings highlight that vitamin D plays an important role in cancer prevention through the FoxO tumor suppressor and coactivator proteins. Another important transcription factor critically involved in cell cycle regulation is c-MYC that is frequently suppressed or overexpressed in cancer. c-MYC is also regulated by 1a,25(OH)2D3. Coimmunoprecipitation studies showed a 1a,25(OH)2D3-dependent association of VDR with c-MYC, suggesting a direct effect of 1a,25(OH)2D3 on c-MYC-regulated cell cycle arrest. However, other data reveal that 1a,25(OH)2D3 exerts both a transcription

123

272

Neuromol Med (2014) 16:265–279

regulatory effect on and enhances turnover of c-MYC (Kim et al. 2003; von der Lehr et al. 2003; Salehi-Tabar et al. 2012; Dimitrov et al. 2013). Vitamin D can modulate b-catenin signaling in cellular homeostasis. The binding of b-catenin to a variety of gene activator or repressor transcription factors may promote or suppress cancer growth. Dysregulation of the Wnt signaling pathway by b-catenin either in the cytoplasm or the nucleus plays important roles in the development of several types of cancer. 1a,25(OH)2D3 interferes with b-catenin signaling by the induction of CDH1 (E-cadherin), which by binding to and retaining b-catenin in the cytoplasm interferes with its regulatory function (Shah et al. 2003; Palmer et al. 2001; Morin 1999). Independent of E-cadherin, 1a,25(OH)2D3 also enhances biding of VDR to b-catenin and promotes the nuclear export of this complex (Shah et al. 2006). The 1a,25(OH)2D3-bound VDR represses bcatenin function, but in contrast, b-catenin can also act as a coactivator of VDR (Shah et al. 2006). In addition to the b-

catenin pathway, there are other pathways involving direct associations of VDR with transcription factors (Jun family, NF-jB, SMAD3) that regulate gene expression, cell proliferation, apoptosis, and differentiation (reviewed in Dimitrov et al. 2013). While the importance of the above-outlined mechanisms has been emphasized in calcium metabolism and cell cycle/ cancer development, the fine-tuning of these molecular interactions is relevant to the involvement of vitamin D/VDR in cellular homeostasis in general.

Vitamin D: VDR in Inflammatory Demyelination (Table 1) While a vitamin D deficiency-related immune dysregulation may contribute to the development of many different disorders, this form of immune abnormalities received the most scrutiny in autoimmunity. The molecular mechanisms

Table 1 VDR actions negatively affected by vitamin D deficiency in inflammatory demyelination Function

VDR effect

Reference

Immune cell development and differentiation

Supports the development of iNKT, CD8aa cells

Bruce and Cantorna (2011), Cantorna et al. (2012)

Antigen-specific activation and proliferation of T cells

Suppresses antigen-specific T-cell activation and proliferation by IL2 and GMCSF expression;

Alroy et al. (1995), Towers and Freedman (1998), Dimitrov et al. (2013), Kongsbak et al. (2013)

Apoptosis

Enhances the expression of pro-apoptotic molecules (calpain 2, caspase-8-associated protein, Bax) in immune cells; NR4A1 and NR4A4:

Spach et al. (2004) and Achiron et al. (2011)

Balance of TH1, TH17 and TH2, and regulatory T cells

Enhances the anti-inflammatory and decreases the pro-inflammatory T-cell activity by IL10:, IL4:, TGFb:, IL17;, Treg:

Deluca and Cantorna (2001), Chang et al. (2010), Sloka et al. (2011), Joshi et al. (2011), Spanier et al. (2012), Nashold et al. (2013)

B-cell homeostasis

Inhibits proliferation and plasma cell differentiation, promotes apoptosis, regulates gene expression, e.g,. HLADRB1, CD40, CXCR4, and CXCR5

Chen et al. (2007), Disanto et al. (2012a, b, c), Ramagopalan et al. (2009, 2010)

Dendritic cell regulation

Inhibits dendritic cell maturation, differentiation, and MHC II expression; induces IL10 production and apoptosis; induces Treg cells and other T-cell subpopulations; modulates chemokine production; induces peripheral tolerance and prevents autoimmunity

van Etten and Mathieu (2005), Adorini and Penna (2009), Spanier et al. (2012), Nashold et al. (2013), Sanseverino et al. (2013)

Innate immune response and TLR signaling

Modulates the innate immune response by the inhibition of TLR8 expression and its signaling pathway; TLR8-dependent proinflammatory cytokines (TNFa,IL1b);

Li et al. (2013a, b)

NFAT/AP1 transcription;

Migration of immune cells via the blood–brain barrier

SOCS (negative regulator): Inhibits T and monocyte transmigration by CCR6;

Chang et al. (2010), Grishkan et al. (2013)

This table summarizes the vitamin D/VDR immune regulatory effects that in case of vitamin D deficiency may enhance the development of inflammatory demyelination or can be targeted by vitamin D treatment in MS/EAE

123

Neuromol Med (2014) 16:265–279

through which VDR regulates the functions of immune cells are highly complex, and the final effects are often dependent of the actual conditions in the experimental systems. It appears that VDR expression does not influence the development and differentiation of CD4?, CD8?, and naturally occurring CD4? FoxP3? Treg (regulatory) T cells in VDR knockout mice or in mice with VDR-deficient T cells (Yu et al. 2008; Mayne et al. 2011). However, the VDR knockout mice have very vigorous immune response and are prone to autoimmunity, perhaps related to the failure to develop certain (other than CD4? FoxP3? Treg) regulatory T cells including the invariant natural killer T (iNKT) cells (Bruce and Cantorna, 2011). The same group showed that vitamin D and VDR deficiency result in an impaired development and low numbers of iNKT cells, indicating that vitamin D is critical for the early development of the immune system (Cantorna et al. 2012). The iNKT cells probably also represent one of the key cell populations that mediates the beneficial effects of calcitriol in EAE (Cantorna et al. 2012). Proliferation of T cells appears to be mediated by a complex association of TFs with VDR. Specifically, 1a,25(OH)2D3 inhibits T-cell proliferation by the suppression of interleukin-2 (IL2) and granulocyte–macrophage colony-stimulating factor (GMCSF) gene transcription. This is accomplished by the blockade of transcription factor NFAT1/AP1 complex formation and the occupation of their binding sites by the VDR/RXR heterodimer. NFAT1 (nuclear factor of activated T cells) and AP1 (activator protein-1) regulate IL2 gene transcription via a 40-kb DNA segment representing the binding site for this complex. VDR without RXR is also inhibiting the NFAT1/AP1 complex formation and DNA binding in a 1a,25(OH)2D3-dependent manner (Alroy et al. 1995; Towers and Freedman 1998; Dimitrov et al. 2013). However, the GMCSF gene promoter is lacking a VDRE sequence. Antigen-specific activation of naı¨ve T cells is associated with the upregulation of VDR expression, and the upregulation of VDRE containing gene of phospholipase C–c1 (PLC-c1) involved in T-cell receptor (TCR) signaling (von Essen et al. 2010). However, proliferation of T cells is driven by IL2, and transcription of IL2 gene is inhibited by VDR (Alroy et al. 1995). Therefore, it is likely that VDR upregulation represents a negative feedback for T-cell-mediated processes and the immune response (reviewed in Kongsbak et al. 2013). Spach et al. (2004) investigated whether 1a,25(OH)2D3 regulates gene expression with an anti-inflammatory outcome. EAE was induced in mice, 1a,25(OH)2D3 or a placebo was injected, and gene expression microarray analyses were performed from the spinal cords 6-h post-injection. While there was no difference in the severity of EAE in the two groups, changes were detected in several 1a,25(OH)2D3-

273

responsive genes. Compared to healthy mice, placebo-treated mice with EAE had an increased expression of many immune inflammatory genes. 1a,25(OH)2D3 treatment caused normalization of several genes whose expression otherwise increased or decreased during EAE. The expressions of two pro-apoptotic gene products, calpain-2 and caspase-8-associated protein, markedly increased. Confirming increased apoptosis in the 1a,25(OH)2D3-treated animals with EAE, a terminal deoxynucleotidyl transferasemediated dUTP nicked end-labeling analysis also showed increased nuclear fragmentation in the CNS lesions. These data suggest that 1a,25(OH)2D3 controls EAE by upregulating pro-apoptotic signals in inflammatory cells. In consensus, Achiron et al. (2011) demonstrated a low expression of the pro-apoptotic nuclear receptor 4A (NR4A) gene family members (NR4A1, NR4A3) and VDR genes in peripheral blood mononuclear cells (PBMC) of subjects with pre-MS or with clinically isolated syndrome (CIS). NR4A1 and NR4A3 are regulated by VDR and primarily involved in T-cell receptor-induced apoptosis. The authors showed that the low T-cell apoptosis in MS patients may be repaired by 1a,25(OH)2D3 primarily through NR4A1 and to some extent through the BCL2-associated X protein (BAX, another proapoptotic molecule). These observations further support the involvement of 1a,25(OH)2D3 and VDR in apoptosis regulation and, through that, the modulation of an eloquent immune cell balance disturbed in autoimmune conditions. Chang et al. (2010) showed that the active form of vitamin D3, 1a,25(OH)2D3, suppresses the differentiation of both the interleukin (IL)-17-producing T cells (T helper-17 or TH17) and the regulatory T cells (Treg). 1a,25(OH)2D3 reduced the amount of IL2 and, through that, the generation of Treg cells, but not TH17 cells. However, under TH17-polarizing conditions, 1a,25(OH)2D3 increased the numbers of IL-10producing T helper-2 (TH2) cells. While the STAT1 signal reciprocally affected the secretion of IL-10 and IL-17, 1a,25(OH)2D3 inhibited IL17 production in STAT1(-/-) T cells. Joshi et al. (2011) showed that in vivo treatment of mice with 1a,25(OH)2D3 during EAE reduces signs of paralysis and disease progression as well as the number of IL-17A-secreting CD4 T cells in the periphery and the CNS. The inhibition of IL-17A expression by 1a,25(OH)2D3 is mediated by VDR through a complex mechanism including the blockade of nuclear factor of activated T cells (NFAT), recruitment of histone deacetylase, sequestration of Runtrelated transcription factor 1 (Runx1) by 1a,25(OH)2D3/ VDR, and a direct effect of 1a,25(OH)2D3 on the induction of Foxp3. Nashold et al. (2013) investigated the effects of vitamin D3 and VDR on regulatory T cells in EAE and found that vitamin D3 alone was ineffective, but the active form, calcitriol? D3, enhanced the presence of Helios?FoxP3? regulatory T cells while decreased the overall number of inflammatory T cells and the T-cell-induced pathology in the

123

274

CNS. The amelioration of pathology was accompanied by decreased clinical severity of EAE. Studies by Deluca and Cantorna (2001) suggest that the immune regulatory action of VDR involves an increased production of IL4 and transforming factor (TGF) b. A study by Sloka et al. (2011) also suggests a shift toward TH2 immune profile induced by 1a,25(OH)2D3, which is enabled by the upregulation of the GATA-3 and STAT6 transcription factors. These studies highlight the modulating effects of vitamin D/VDR on T effector and regulatory cell balances, and suggest new therapeutic targets for the control of EAE/MS. Spanier et al. (2012) found that vitamin D3 failed to inhibit EAE in Ifng knockout (GKO) mice unlike in wildtype mice. The GKO mice cannot produce interferon-c due to the disruption of the Ifng gene. The two strains had similar Cyp27b1 and Cyp24a1 gene expression, suggesting similar vitamin D3 metabolism in the CNS. To inhibit EAE, however, 2 times more 1,25-(OH)2D3 was needed in GKO mice, which also had very low Vdr expression in the CNS. Intracranial injection of IFN-c did not increase Vdr gene expression, and the low Vdr expression was associated with increased pathogenic TH1 and TH17 cells in the CNS of GKO mice. In contrast to the studies of Nashold et al. (2013), in this study, 1,25-(OH)2D3 reduced the numbers of the TH1 and TH17 cells in GKO and wild-type mice without altering Foxp3? regulatory T cells (Spanier et al. 2012). Thus, the Ifng gene was necessary for Vdr expression, regulation of T-cell subpopulations, and mediation of a vitamin D3-dependent mechanism that inhibits EAE. This study is relevant to MS in Sardinian patients who frequently carry an Ifng allele associated with low protein expression. This ethnic population has relatively high MS prevalence despite strong UV irradiation. The studies by Spanier et al. (2012) and Nashold et al. (2013) complement the observations by Adorini and Penna (2009) demonstrating that VDR can induce tolerogenic dendritic cells capable of promoting CD4? CD25? Foxp3? suppressor T cells and thus regulating the balances of T effector cells. The modulating effects of dendritic cells on T cells may be mediated, at least in part, by chemokines whose expressions are regulated by VDR (Sanseverino et al. 2013). In addition, direct actions of 1,25-(OH)2D3 on dendritic cells include the inhibition of differentiation, maturation, and MHC II expression and the enhancement of IL10 production and apoptosis (van Etten and Mathieu 2005). 1a,25(OH)2D3 treatment not only modulates the distribution and function of T-cell subpopulations, but also renders T-helper cells unable to enter into the CNS from the peripheral circulation (Grishkan et al. 2013). This inhibitory effect may be related to a negative regulation of CC chemokine receptors (CCRs), particularly of CCR6, needed for the entry of pathogenic T lymphocytes such as

123

Neuromol Med (2014) 16:265–279

TH17 cells in the CNS and for the initiation of EAE (Chang et al. 2010). Complementarily, chemokines expressed in the CNS play important roles in attracting the transmigration of activated immune cells equipped with appropriate CCRs into the CNS. In a study by Sanseverino et al. (2013), both 1,25(OH)2D3 and 25(OH)D3 appeared to induce increased secretion of CCL2. The 1,25(OH)2D3induced CC chemokine ligand (CCL)-2 levels were similar to those induced by classical dendritic cell maturation stimuli. While 1,25(OH)2D3 also induced CCL2 secretion in dendritic cells of patients with relapsing-remitting MS, the levels of patients cell-derived CCL2 were lower than those obtained from dendritic cells of healthy controls. The causes and mechanisms of this deficiency were unclear, but were likely related to the complex immune dysregulation observed in MS. The effects of vitamin D/VDR on B cells are broad and only partly elucidated. Studies in B-cell lines (Ramagopalan et al. 2009, 2010; Disanto et al. 2012a, b, c) point out that VDR has binding sites within, and thus, vitamin D regulates the expression of many genes that carry variants associated with MS susceptibility. Products of the VDRregulated HLA-DRB1, CD40, CXCR4, and CXCR5 genes allow B cells to participate in antigen presentation, T-cell activation, and differentiation as well as responding to chemotactic signals (Ramagopalan et al. 2009, Disanto et al. 2012c). However, vitamin D/VDR also inhibit proliferation and differentiation of B cells, while enhance apoptosis (Diasanto et al. 2012c, Chen et al. 2007). Li et al. (2013a) investigated the effect of 1a,25(OH)2D3 and Toll-like receptor (TLR)7/8 signaling. TLRs recognize pathogen-associated molecular patterns (PAMPs) of various microbial molecules that may cause an overactivation of the TLR-expressing cells of the innate immune system and a heightened production of proinflammatory cytokines implicated in autoimmune conditions including MS and EAE. 1a,25(OH)2D3 injected prior to or after the induction of EAE reduced the production of inflammatory cytokines, expression of several TLRs (most strikingly of TLR8) and the severity of EAE. 1a,25(OH)2D3 significantly reduced TLR8 expression through transcription regulation and also diminished the expression or activity of MyD88, IRF-4, IRF-7, and NF-kB in monocytes exposed to TLR8 ligands. By interfering with the TLR8 gene and its signaling pathway, 1a,25(OH)2D3 reduced the expression of the TLR8 proinflammatory cytokine targets, TNF-a, and IL-1b. These observations (Li et al. 2013a) could also explain the findings by Nashold et al. (2000) describing an EAE suppressing effect of 1a,25(OH)2D3 in association with reduced numbers of inflammatory monocytes/macrophages in the CNS. However, it is important to note that vitamin D signaling induces IL1B gene expression in human macrophages by a mechanism that is not conserved in mouse

Neuromol Med (2014) 16:265–279

(Verway et al. 2013). Another study group (Li et al. 2013b) investigated the involvement of SOCS proteins in TLR signaling in relation to the VDR-mediated regulation. The SOCS protein family is involved in a negative feedback loop that regulates several aspects of cytokine signaling. miR-155 is a regulator of TLR signaling that targets SOCS1 and blocks the negative feedback loop in activated macrophages. This study showed that 1a,25(OH)2D3 is able to modulate the innate immune response through the involvement of the miR-155-SOCS1 axis. Vdr deletion caused enhanced inflammatory response in mice and in cultures of macrophages stimulated by lipopolysaccharide, because of the overproduction of miR-155 that suppresses SOCS1. In mice with bic/miR-155 deletion, 1a,25(OH)2D3 suppressed inflammation and stimulated SOCS1. 1a,25(OH)2D3 downregulated bic transcription by blocking NF-jB activation, and VDR inhibited NF-jB activation by interacting with IKKb. This study reveals that VDR signaling reduces TLR-mediated inflammation by enhancing the negative feedback loop. Thus, the above two studies expand our knowledge on the immune regulatory effect of 1a,25(OH)2D3 and show that not only the adaptive but also the innate immune system may be affected by the vitamin D/VDR signaling pathway. In a study by Eyles et al. (2007), vitamin D-deficient female rats were mated with non-deficient males and the offspring were fed with a regular diet. Gene expression in the brains of 10-week-old offspring was compared with that of controls using Affymetrix microarrays. Prenatal D hypovitaminosis caused dysregulation of several key pathways including oxidative phosphorylation, cytoskeletal maintenance, calcium homeostasis, posttranslational regulation, synaptic development, and neurotransmission. Since abnormalities in mitochondrial biochemical processes has been associated with neurodegeneration in MS and synaptic development abnormalities in schizophrenia, the authors postulate that prenatal vitamin D deficiency may underlie the development of both diseases. Protein–protein interaction studies confirm and reconcile data of the above-reviewed papers (Tuller et al. 2013). Chemokines including CXCL1-3, 5, 6, and IL8 showed a tendency for differential expression in PBMNC in various autoimmune diseases, and the differentially expressed genes physically interacted with the anti-apoptotic gene BCL3, interferon-c, and VDR genes. However, each autoimmune disease appeared to be related to distinct cellular processes and distinct sets of differentially expressed genes. The cellular processes related to cell proliferation (e.g., growth factors, NFjB, Wnt/b-catenin signaling, and stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (e.g., IL2, IL6, GMCSF, and B-cell receptor), general signaling cascades (e.g., various kinases and signaling molecules), and apoptosis were found to be activated

275

in most of the studied autoimmune diseases, but in each one of them, apoptosis and chemotaxis were found to be activated via different subsignaling pathways (Tuller et al. 2013). VDR is involved in many conditions and complex cellular processes, but the disease-relevant processes are quite distinct in various conditions and diseases. Studying the interaction of VDR with the genome and its involvement in gene expression regulation has greatly facilitated the improvement of our understanding as to how inflammatory demyelination develops under the control of an environmental factor.

Conclusions These studies highlight the complexity of mechanisms underlying the pleiotropic biological effects of vitamin D/VDR and identify specific levels of molecular interactions through which vitamin D/VDR deficiency may influence the development of inflammatory demyelination in the CNS. The best defined and most comprehensive information is derived from genome-wide Chip-seq analyses combined with transcriptome studies, which reveal the exact sites, gene-related positions, specific DNA sequences, and molecular complexity as well as transcriptional consequences of VDR–genomic interactions. The involvements of specific and unspecific VDREs in the proximity of gene TSSs, active promoters and strong enhancers, and the varying interactions of VDR with multitudes of other nuclear factors, epigenetic regulators, and signaling molecules explain, at least in part, the complexity of final vitamin D effects in various tissues and conditions. MS is one of the best characterized conditions in which vitamin D acts as a significant environmental factor. The data show that the majority of VDR-binding sites involves genes of immune regulation and overlaps with MS susceptibility SNP variants identified genomewide. Functional studies also reveal that apoptosis, balances in T helper and regulatory subpopulations, iNKT, dendritic and B-cell maturation, TLR and MMP expressions, cytokine and chemokine interactions with their relevant receptors, immune cell transmigration via the blood– brain barrier as well as mitochondrial energy metabolism may be among those key processes that are modulated by vitamin D levels in inflammatory demyelination. The sum of VDR–ligand immune effects includes a suppression of pro-inflammatory and enhancement of anti-inflammatory processes. Deficiency in the vitamin D supply thus may facilitate a proinflammatory shift and lead to autoimmunity in an appropriate genetic background. There is now enough scientific evidence to justify the utilization of vitamin D as a therapeutic modality in MS; however, further clinical studies are needed to define the specifics of such

123

276

Neuromol Med (2014) 16:265–279

intervention. Vitamin D certainly represents an important environmental factor that interacts with the genome and influences gene expression regulation in such a way that aberrations in it can facilitate the development of complex trait diseases. Manipulations of this gene–environmental interactions not only improves our understanding of immune dysregulation, but may also promote the development of a strategy that can first in history prevent or ameliorate the risk of autoimmunity. Acknowledgments The authors are supported by the Markusovszky University Teaching Hospital and the University of Pecs, Faculty of the Health Sciences. Conflict of interest of interests.

The authors declare that they have no conflict

References Achiron, A., Feldman, A., & Gurevich, M. (2011). Characterization of multiple sclerosis traits: Nuclear receptors (NR) impaired apoptosis pathway and the role of 1-a 25-dihydroxyvitamin D3. Journal of the Neurological Sciences, 311(1–2), 9–14. Adorini, L., & Penna, G. (2009). Induction of tolerogenic dendritic cells by vitamin D receptor agonists. Handbook of Experimental Pharmacology, 188, 251–273. Adzemovic, M. Z., Zeitelhofer, M., Hochmeister, S., Gustafsson, S. A., & Jagodic, M. (2013). Efficacy of vitamin D in treating multiple sclerosis-like neuroinflammation depends on developmental stage. Experimental Neurology, 249, 39–48. Alroy, I., Towers, T. L., & Freedman, L. P. (1995). Transcriptional repression of the interleukin-2 gene by vitamin D3: Direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Molecular and Cellular Biology, 15(10), 5789–5799. An, B. S., Tavera-Mendoza, L. E., Dimitrov, V., Wang, X., Calderon, M. R., Wang, H. J., et al. (2010). Stimulation of Sirt1-regulated FoxO protein function by the ligand-bound vitamin D receptor. Molecular and Cellular Biology, 30(20), 4890–4900. Ascherio, A., & Munger, K. L. (2010). Epstein-barr virus infection and multiple sclerosis: A review. J Neuroimmune Pharmacol., 5(3), 271–277. Ascherio, A., Munger, K. L., & Simon, K. C. (2010). Vitamin D and multiple sclerosis: Review. The Lancet Neurology, 9(6), 599–612. Aslam, F., McCabe, L., Frenkel, B., van Wijnen, A. J., Stein, G. S., Lian, J. B., et al. (1999). AP-1 and vitamin D receptor (VDR) signaling pathways converge at the rat osteocalcin VDR element: Requirement for the internal activating protein-1 site for vitamin D-mediated trans-activation. Endocrinology, 140(1), 63–70. Baranzini, S. E. (2011). Revealing the genetic basis of multiple sclerosis: Are we there yet? Current Opinion in Genetics & Development, 21(3), 317–324. Baranzini, S. E., Galwey, N. W., Wang, J., Khankhanian, P., Lindberg, R., Pelletier, D., Wu, W., Uitdehaag, B. M., Kappos, L., GeneMSA Consortium, Polman, C. H., Matthews, P. M., Hauser, S. L., Gibson, R. A., Oksenberg, J. R., Barnes, M. R. (2009). Pathway and network-based analysis of genome-wide association studies in multiple sclerosis. Human Molecular Genetics, 18(11), 2078–2090.

123

Barreyro, F. J., Kobayashi, S., Bronk, S. F., Werneburg, N. W., Malhi, H., & Gores, G. J. (2007). Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis. Journal of Biological Chemistry, 282(37), 27141–27154. Bernstein, B. E., Birney, E., Dunham, I., Green, E. D., Gunter, C., & Snyder, M. (2012). ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature, 489(7414), 57–74. Brennan, A., Katz, D. R., Nunn, J. D., Barker, S., Hewison, M., Fraher, L. J., et al. (1987). Dendritic cells from human tissues express receptors for the immunoregulatory vitamin D3 metabolite, dihydroxycholecalciferol. Immunology, 61(4), 457–461. Bruce, D., & Cantorna, M. T. (2011). Intrinsic requirement for the vitamin D receptor in the development of CD8aa-expressing T cells. J Immunol., 186(5), 2819–2825. Cantorna, M. T., Hayes, C. E., & DeLuca, H. F. (1996). 1,25Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America, 93(15), 7861–7864. Cantorna, M. T., Zhao, J., & Yang, L. (2012). Vitamin D, invariant natural killer T-cells and experimental autoimmune disease. The Proceedings of the Nutrition Society, 71(1), 62–66. Chang, J. H., Cha, H. R., Lee, D. S., Seo, K. Y., & Kweon, M. N. (2010). 1,25-Dihydroxyvitamin D3 inhibits the differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis. PLoS ONE, 5(9), e12925. Chen, S., Sims, G. P., Chen, X. X., Gu, Y. Y., & Lipsky, P. E. (2007). Modulatory effects of 1,3-dihydroxyvitamin D3 on human B cell differentiation. Journal of Immunology, 179, 1634–1647. Compston, A. (1999). The genetic epidemiology of multiple sclerosis. Philosophical Transactions of the Royal Society of London. Series B, Biological sciences, 354(1390), 1623–1634. Daitoku, H., Hatta, M., Matsuzaki, H., Aratani, S., Ohshima, T., Miyagishi, M., et al. (2004). Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proceedings of the National Academy of Sciences of the United States of America, 101(27), 10042–10047. Deluca, H. F., & Cantorna, M. T. (2001). Vitamin D: Its role and uses in immunology. FASEB Journal, 15(14), 2579–2585. Dimitrov, V., Salehi-Tabar, R., An, B. S., & White, J. H. (2013). Nonclassical mechanisms of transcriptional regulation by the vitamin D receptor: Insights into calcium homeostasis, immune system regulation and cancer chemoprevention. The Journal of Steroid Biochemistry and Molecular Biology. doi:10.1016/j.jsbmb.2013. 07.012. Disanto, G., Morahan, J. M., Barnett, M. H., Giovannoni, G., & Ramagopalan, S. V. (2012a). The evidence for a role of B cells in multiple sclerosis. Neurology, 78(11), 823–832. Disanto, G., Sandve, G. K., Berlanga-Taylor, A. J., Morahan, J. M., Dobson, R., Giovannoni, G., et al. (2012b). Genomic regions associated with multiple sclerosis are active in B cells. PLoS ONE, 7(3), e32281. doi:10.1371/journal.pone.0032281. Disanto, G., Sandve, G. K., Berlanga-Taylor, A. J., Ragnedda, G., Morahan, J. M., Watson, C. T., et al. (2012c). Vitamin D receptor binding, chromatin states and association with multiple sclerosis. Human Molecular Genetics, 21(16), 3575–3586. Do¨rr, J., Do¨ring, A., & Paul, F. (2013). Can we prevent or treat multiple sclerosis by individualised vitamin D supply? EPMA Journal, 4(1), 4. doi:10.0086/1878-5085-4-4. Do¨rr, J., Ohlraun, S., Skarabis, H., & Paul, F. (2012). Efficacy of vitamin D supplementation in multiple sclerosis (EVIDIMS Trial): Study protocol for a randomized controlled trial. Trials, 13, 15. Ernst, J., Kheradpour, P., Mikkelsen, T. S., Shoresh, N., Ward, L. D., Epstein, C. B., et al. (2011). Mapping and analysis of chromatin

Neuromol Med (2014) 16:265–279 state dynamics in nine human cell types. Nature, 473(7345), 43–49. Eyles, D., Almeras, L., Benech, P., Patatian, A., Mackay-Sim, A., McGrath, J., et al. (2007). Developmental vitamin D deficiency alters the expression of genes encoding mitochondrial, cytoskeletal and synaptic proteins in the adult rat brain. Journal of Steroid Biochemistry and Molecular Biology, 103(3–5), 538–545. Garcia-Martin, E., Agundez, J. A., Martinez, C., Benito-Leon, J., Millan-Pacual, J., Calleja, P., et al. (2013). Vitamin D3 receptor (VDR) gene rs2228570 (Fok1) and rs731236 (Taq1) variants are not associated with the risk for multiple sclerosis: Results of a new study and a meta-analysis. PLoS ONE, 8(6), e65487. doi:10. 1371/journal.pone.0065487. Grishkan, I. V., Fairchild, A. N., Calabresi, P. A., & Gocke, A. R. (2013). 1,25-Dihydroxyvitamin D3 selectively and reversibly impairs T helper-cell CNS localization. Proceedings of the National Academy of Sciences of the United States of America, 110(52), 21101–21106. Hafler, D. A., Compston, A., Sawcer, S., Lander, E. S., Daly, M. J., De Jager, P. L., et al. (2007). Risk alleles for multiple sclerosis identified by a genomewide study. New England Journal of Medicine, 357, 851–862. Handel, A. E., Sandve, G. K., Disanto, G., Berlanga-Taylor, A. J., Gallone, G., Hanwell, H., et al. (2013). Vitamin D receptor ChIP-seq in primary CD4? cells: Relationship to serum 25-hydroxyvitamin D levels and autoimmune disease. BMC Medicine, 11, 163. doi:10.1186/1741-7015-11-163. Handunnetthi, L., Ramagopalan, S. V., & Ebers, G. C. (2010). Multiple sclerosis, vitamin D, and HLA-DRB1*15. Neurolog., 74(23), 1905–1910. doi:10.1212/WNL.0b013e3181e24124. Heikkinen, S., Va¨isa¨nen, S., Pehkonen, P., Seuter, S., Benes, V., & Carlberg, C. (2011). Nuclear hormone 1a,25-dihydroxyvitamin D3 elicits a genome-wide shift in the locations of VDR chromatin occupancy. Nucleic Acids Research, 39(21), 9181–9193. James, E., Dobson, R., Kuhle, J., Baker, D., Giovannoni, G., & Ramagopalan, S. V. (2013). The effect of vitamin D-related interventions on multiple sclerosis relapses: A meta-analysis. Multiple Sclerosis, 19(12), 1571–1579. Joshi, S., Pantalena, L. C., Liu, X. K., Gaffen, S. L., Liu, H., Rohowsky-Kochan, C., et al. (2011). 1,25-dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcriptional modulation of interleukin-17A. Molecular and Cellular Biology, 31(17), 3653–3669. Kim, M. S., Fujiki, R., Murayama, A., Kitagawa, H., Yamaoka, K., Yamamoto, Y., et al. (2007). 1Alpha,25(OH)2D3-induced transrepression by vitamin D receptor through E-box-type elements in the human parathyroid hormone gene promoter. Molecular Endocrinology, 21(2), 334–342. Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E., & Tansey, W. P. (2003). Skp2 regulates Myc protein stability and activity. Molecular Cell, 11(5), 1177–1188. Knippenberg, S., Smolders, J., Thewissen, M., Peelen, E., Tervaert, J. W., Hupperts, R., et al. (2011). Effect of vitamin D(3) supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis. Multiple Sclerosis, 17(12), 1418–1423. Kongsbak, M., Levring, T. B., Geisler, C., & von Essen, M. R. (2013). The vitamin d receptor and T cell function. Frontiers in Immunology, 4, 148. doi:10.3389/fimmu.2013.00148. Li, B., Baylink, D. J., Deb, C., Zannetti, C., Rajaallah, F., Xing, W., et al. (2013a). 1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo. PLoS ONE, 8(3), e58808. doi:10.1371/journal.pone.0058808.

277 Li, Y. C., Chen, Y., Liu, W., & Thadhani, R. (2013b). MicroRNAmediated mechanism of vitamin D regulation of innate immune response. The Journal of Steroid Biochemistry and Molecular Biology. doi: 10.1016/j.jsbmb.2013.09.014. Lin, R., Wang, T. T., Miller, W. H, Jr, & White, J. H. (2003). Inhibition of F-Box protein p45(SKP2) expression and stabilization of cyclin-dependent kinase inhibitor p27(KIP1) in vitamin D analog-treated cancer cells. Endocrinology, 144(3), 749–753. Mayne, C. G., Spanier, J. A., Relland, L. M., Williams, C. B., & Hayes, C. E. (2011). 1,25-Dihydroxyvitamin D3 acts directly on the T lymphocyte vitamin D receptor to inhibit experimental autoimmune encephalomyelitis. European Journal of Immunology, 41(3), 822–832. Meyer, M. B., Benkusky, N. A., & Pike, J. W. (2013). 1,25Dihydroxyvitamin D3 induced histone profiles guide discovery of VDR action sites. The Journal of Steroid Biochemistry and Molecular Biology. doi:10.1016/j.jsbmb.2013.09.005. Mora´n-Auth, Y., Penna-Martinez, M., Shoghi, F., Ramos-Lopez, E., & Badenhoop, K. (2013). Vitamin D status and gene transcription in immune cells. Journal of Steroid Biochemistry and Molecular Biology, 136, 83–85. doi:10.1016/j.jsbmb.2013.02. 005. Morin, P. J. (1999). Beta-catenin signaling and cancer. BioEssays, 21(12), 1021–1030. Murayama, A., Kim, M. S., Yanagisawa, J., Takeyama, K., & Kato, S. (2004). Transrepression by a liganded nuclear receptor via a bHLH activator through co-regulator switching. EMBO Journal, 23(7), 1598–1608. Nashold, F. E., Miller, D. J., & Hayes, C. E. (2000). 1,25dihydroxyvitamin D3 treatment decreases macrophage accumulation in the CNS of mice with experimental autoimmune encephalomyelitis. Journal of Neuroimmunology, 103(2), 171–179. Nashold, F. E., Nelson, C. D., Brown, L. M., & Hayes, C. E. (2013). One calcitriol dose transiently increases Helios? FoxP3? T cells and ameliorates autoimmune demyelinating disease. Journal of Neuroimmunology, 263(1–2), 64–74. Palmer, H. G., Gonza´lez-Sancho, J. M., Espada, J., Berciano, M. T., Puig, I., Baulida, J., et al. (2001). Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. Journal of Cell Biology, 154(2), 369–387. Ramagopalan, S. V., Dyment, D. A., Cader, M. Z., Morrison, K. M., Disanto, G., Morahan, J. M., et al. (2011). Rare variants in the CYP27B1 gene are associated with multiple sclerosis. Annals of Neurology, 70(6), 881–886. Ramagopalan, S. V., Heger, A., Berlanga, A. J., Maugeri, N. J., Lincoln, M. R., Burrell, A., et al. (2010). A ChIP-seq defined genome-wide map of vitamin D receptor binding: Associations with disease and evolution. Genome Research, 20(10), 1352–1360. Ramagopalan, S. V., Lee, J. D., Yee, I. M., Guimond, C., Traboulsee, A. L., Ebers, G. C., et al. (2013). Association of smoking with risk of multiple sclerosis: A population-based study. Journal of Neurology, 260(7), 1778–1781. Ramagopalan, S. V., Maugeri, N. J., Handunnetthi, L., Lincoln, M. R., Orton, S. M., Dyment, D. A., et al. (2009). Expression of the multiple sclerosis-associated MHC class II Allele HLADRB1*1501 is regulated by vitamin D. PLoS Genetics, 5(2), e1000369. doi:10.1371/journal.pgen.1000369. Salehi-Tabar, R., Nguyen-Yamamoto, L., Tavera-Mendoza, L. E., Quail, T., Dimitrov, V., An, B. S., et al. (2012). Vitamin D receptor as a master regulator of the c-MYC/MXD1 network. Proceedings of the National Academy of Sciences of the United States of America, 109(46), 18827–18832.

123

278 Salzer, J., & Sundstro¨m, P. (2013). Timing of cigarette smoking as a risk factor for multiple sclerosis. Therapeutic Advances in Neurological Disorders, 6(3), 205. Sanseverino, I., Rinaldi, A. O., Purificato, C., Cortese, A., Millefiorini, E., Gessani, S., & Gauzzi, M. C. (2013). CCL2 induction by 1,25(OH)2D3 in dendritic cells from healthy donors and multiple sclerosis patients. The Journal of Steroid Biochemistry and Molecular Biology. doi:10.1016/j.jsbmb.2013.10.018. Satoh, J., & Tabunoki, H. (2013). Molecular network of chromatin immunoprecipitation followed by deep sequencing-based vitamin D receptor target genes. Multiple Sclerosis, 19(8), 1035–1045. Schmidt, M., Fernandez de Mattos, S., van der Horst, A., Klompmaker, R., Kops, G. J., Lam, E. W., et al. (2002). Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Molecular and Cellular Biology, 22(22), 7842–7852. Seuter, S., Heikkinen, S., & Carlberg, C. (2013). Chromatin acetylation at transcription start sites and vitamin D receptor binding regions relates to effects of 1a,25-dihydroxyvitamin D3 and histone deacetylase inhibitors on gene expression. Nucleic Acids Research, 41(1), 110–124. Shah, S., Hecht, A., Pestell, R., & Byers, S. W. (2003). Transrepression of beta-catenin activity by nuclear receptors. Journal of Biological Chemistry, 278(48), 48137–48145. Shah, S., Islam, M. N., Dakshanamurthy, S., Rizvi, I., Rao, M., Herrell, R., et al. (2006). The molecular basis of vitamin D receptor and beta-catenin crossregulation. Molecular Cell, 21(6), 799–809. Simon, K. C., Munger, K. L., & Ascherio, A. (2012). Vitamin D and multiple sclerosis: Epidemiology, immunology, and genetics. Current Opinion in Neurology, 25(3), 246–251. Simon, K. C., Munger, K. L., Yang, Xing, & Ascherio, A. (2010a). Polymorphisms in vitamin D metabolism related genes and risk of multiple sclerosis. Multiple Sclerosis, 16(2), 133–138. Simon, K. C., van der Mei, I. A., Munger, K. L., Ponsonby, A., Dickinson, J., Dwyer, T., et al. (2010b). Combined effects of smoking, anti-EBNA antibodies, and HLA-DRB1*1501 on multiple sclerosis risk. Neurology., 74(17), 1365–1371. Sloka, S., Silva, C., Wang, J., & Yong, V. W. (2011). Predominance of Th2 polarization by vitamin D through a STAT6-dependent mechanism. J Neuroinflammation., 8, 56. doi:10.1186/17422094-8-56. Smolders, J., & Damoiseaux, J. (2011). Vitamin D as a T-cell modulator in multiple sclerosis. Vitamins and Hormones, 86, 401–428. Smolders, J., Damoiseaux, J., Menheere, P., Tervaert, J. W., & Hupperts, R. (2009). Association study on two vitamin D receptor gene polymorphisms and vitamin D metabolites in multiple sclerosis. Annals of the New York Academy of Sciences, 1173, 515–520. doi:10.1111/j.1749-6632.2009.04656.x. Smolders, J., Hupperts, R., Barkhof, F., Grimaldi, L. M., Holmoy, T., Killestein, J., et al. (2011). Efficacy of vitamin D3 as add-on therapy in patients with relapsing-remitting multiple sclerosis receiving subcutaneous interferon b-1a: A Phase II, multicenter, double-blind, randomized, placebo-controlled trial. Journal of the Neurological Sciences, 311(1–2), 44–49. Smolders, J., Peelen, E., Thewissen, M., Menheere, P., Damoiseaux, J., & Hupperts, R. (2014). Circulating vitamin D binding protein levels are not associated with relapses or with vitamin D status in multiple sclerosis. Multiple Sclerosis, 20(4), 433–437. Smolders, J., Schuurman, K. G., van Strien, M. E., Melief, J., Hendrickx, D., Hol, E. M., et al. (2013). Expression of vitamin D receptor and metabolizing enzymes in multiple sclerosis-affected brain tissue. Journal of Neuropathology and Experimental Neurology, 72(2), 91–105.

123

Neuromol Med (2014) 16:265–279 Spach, K. M., Pedersen, L. B., Nashold, F. E., Kayo, T., Yandell, B. S., Prolla, T. A., et al. (2004). Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis. Physiological Genomics, 18(2), 141–151. Spanier, J. A., Nashold, F. E., Olson, J. K., & Hayes, C. E. (2012). The Ifng gene is essential for Vdr gene expression and vitamin D3-mediated reduction of the pathogenic T cell burden in the central nervous system in experimental autoimmune encephalomyelitis, a multiple sclerosis model. Journal of Immunology, 189(6), 3188–3197. Tajouri, L., Ovcaric, M., Curtain, R., Johnson, M. P., Griffiths, L. R., Csurhes, P., et al. (2005). Variation in the vitamin D receptor gene is associated with multiple sclerosis in an Australian population. Journal of Neurogenetics, 19(1), 25–38. The International Multiple Sclerosis Consortium. (2013b). Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis. Nature Genetics, 45(11), 1353–1360. The International Multiple Sclerosis Consortium and the Wellcome Trust Case Control Consortium 2. (2011). Genetic risk and a primary role for cell-mediated immune mechanism in multiple sclerosis. Nature, 476, 214–218. The International Multiple Sclerosis Genetics Consortium. (2013a). Network-based multiple sclerosis pathway analysis with GWAS data from 15,000 cases and 30,000 controls. American Journal of Human Genetics, 92, 1–12. Towers, T. L., & Freedman, L. P. (1998). Granulocyte-macrophage colony-stimulating factor gene transcription is directly repressed by the vitamin D3 receptor. Implications for allosteric influences on nuclear receptor structure and function by a DNA element. Journal of Biological Chemistry, 273(17), 10338–10348. Tuller, T., Atar, S., Ruppin, E., Gurevich, M., & Achiron, A. (2013). Common and specific signatures of gene expression and protein– protein interactions in autoimmune diseases. Genes and Immunity, 14(2), 67–82. van Etten, E., & Mathieu, C. (2005). Immunoregulation by 1,25dihydroxyvitamin D3: Basic concepts. The Journal of Steroid Biochemistry and Molecular Biology, 97(1–2), 93–101. Verstuyf, A., Carmeliet, G., Bouillon, R., & Mathieu, C. (2010). Vitamin D: A pleiotropic hormone. Kidney International, 78, 140–145. Verway, M., Bouttier, M., Wang, T. T., Calderon, M., An, B. S., Devemy, E., Mcintosh, F., Divangahi, M., Behr, M. A., & White, J. H. (2013). Vitamin D induces interleukin-1b expression: Paracrine macrophage epithelial signaling controls M. tuberculosis infection. PLOS Pathogens. doi:10.1371/journal.ppat. 1003407. Virtanen, J. O., & Jacobson, S. (2012). Viruses and multiple sclerosis. CNS & Neurological Disorders: Drug Targets, 11(5), 528–544. von der Lehr, N., Johansson, S., Wu, S., Bahram, F., Castell, A., Cetinkaya, C., et al. (2003). The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. Molecular Cell, 11(5), 1189–1200. von Essen, M. R., Kongsbak, M., Schjerling, P., Olgaard, K., Odum, N., & Geisler, C. (2010). Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nature Immunology, 11(4), 344–349. Willer, C. J., Dyment, D. A., Risch, N. J., Sadovnick, A. D., & Ebers, G. C. (2003). Twin concordance and sibling recurrence rates in multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America, 100(22), 12877– 12882.

Neuromol Med (2014) 16:265–279 Yu, S., Bruce, D., Froicu, M., Weaver, V., & Cantorna, M. T. (2008). Failure of T cell homing, reduced CD4/CD8alphaalpha intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice. Proceedings of the National Academy of Sciences of the United States of America, 105(52), 20834–20839.

279 Zittermann, A., Tenderich, G., & Koerfer, R. (2009). Vitamin D and the adaptive immune system with special emphasis to allergic reactions and allograft rejection. Inflammation & Allergy: Drug Targets, 8(2), 161–168.

123

Genomic binding sites and biological effects of the vitamin D--VDR complex in multiple sclerosis [corrected].

Environmental factors greatly contribute to the development of complex trait disorders. With the rapid developments in the fields of biotechnology and...
585KB Sizes 0 Downloads 3 Views