Ainst the respective change of the IL6 protein concentrations. doi:10.1371/journal.pone.0071042.g155.7 nM and ii) changes ranging from a decrease by 30.2 nM and an increase by 87.2 nM. In contrast to most other studies reported, we express these changes in relative and not in absolute terms, i.e. as a ratio and not as a difference. Therefore, we interpret the changes in serum 25(OH)D3 concentrations, which were achieved by the VitDmet study, as a range from a 2.1-fold decrease of the baseline levels up to a 2.8-fold increase. In this way, our approach is closer to the analysis of a typical ligand stimulation experiment as it is the standard in mechanistic studies [39]. Accordingly, the mRNA expression changes range in PBMCs from a 1.8-fold decrease to a 1.9-fold increase for CD14, from a 2.0-fold decrease to a 1.9-fold increase for THBD and from a 1.8fold decrease to a 1.9-fold increase for VDR. In adipose tissue samples the ranges in mRNA expression changes are even larger spanning from a 3.4-fold decrease to a 2.6-fold increase for CD14, from a 4.8-fold decrease to a 2.9-fold increase for THBD and from a 4.0-fold decrease to a 4.1-fold increase for VDR. Interestingly, Title Loaded From File although VDR expression changes do not correlate with changes in 25(OH)D3 serum concentrations, the VDR gene shows similar ranges of variation than CD14 and THBD. Although the ranges of the 25(OH)D3 serum concentration and VDR target gene changes during the intervention are in the same order, there is no statistically significant correlation between them, when all 71 study participants are studied. However, after ranking the study participants by the responsiveness of their CD14 and THBD expression to changes of 25(OH)D3 concentrations in both tested tissues, we found in the top half of the ranked participants a significant positive correlation. From the latter 35 individuals only 3 Title Loaded From File showed a slight decrease in 25(OH)D3 concentrations, i.e. majority of them seem to benefit from the intervention irrespective of their initial serum 25(OH)D3 concentration. In fact, only 4 of the 35 participants had an initial 25(OH)D3 concentration of below 50 nM, i.e. according to the recent IoM recommendations [6] most of the participants would not have needed a vitamin D supplementation. For the other half of the study group no relationship between changes in 25(OH)D3 concentrations and VDR target gene expression could be found. These individuals showed a more individual response to vitamin D supplementation (or the lack of it) and no general conclusion could be reached from gene expression data.We suggest that analysis of the responsiveness of the genes CD14 and THBD to changes in 25(OH)D3 serum concentrations allows a categorization of the study participants. Half of the participants can be considered as conventional responders to vitamin D. These individuals have a fully functional vitamin D signaling system and their vitamin D concentrations have not reached saturation. This is proven by the down-regulation of IL6 protein in serum. IL6 has not yet been shown to be a primary VDR target, but it is known as one of the genes, via which the anti-inflammatory effect 23977191 of vitamin D is mediated [42,43]. IL6 is a marker of low-grade inflammation and has been suggested as a risk factor for type 2 diabetes [44] and cardiovascular disease [45]. Therefore, the down-regulation of IL6 protein in response to raising 25(OH)D3 serum concentrations is an indication of a beneficial effect of vitamin D3 sup.Ainst the respective change of the IL6 protein concentrations. doi:10.1371/journal.pone.0071042.g155.7 nM and ii) changes ranging from a decrease by 30.2 nM and an increase by 87.2 nM. In contrast to most other studies reported, we express these changes in relative and not in absolute terms, i.e. as a ratio and not as a difference. Therefore, we interpret the changes in serum 25(OH)D3 concentrations, which were achieved by the VitDmet study, as a range from a 2.1-fold decrease of the baseline levels up to a 2.8-fold increase. In this way, our approach is closer to the analysis of a typical ligand stimulation experiment as it is the standard in mechanistic studies [39]. Accordingly, the mRNA expression changes range in PBMCs from a 1.8-fold decrease to a 1.9-fold increase for CD14, from a 2.0-fold decrease to a 1.9-fold increase for THBD and from a 1.8fold decrease to a 1.9-fold increase for VDR. In adipose tissue samples the ranges in mRNA expression changes are even larger spanning from a 3.4-fold decrease to a 2.6-fold increase for CD14, from a 4.8-fold decrease to a 2.9-fold increase for THBD and from a 4.0-fold decrease to a 4.1-fold increase for VDR. Interestingly, although VDR expression changes do not correlate with changes in 25(OH)D3 serum concentrations, the VDR gene shows similar ranges of variation than CD14 and THBD. Although the ranges of the 25(OH)D3 serum concentration and VDR target gene changes during the intervention are in the same order, there is no statistically significant correlation between them, when all 71 study participants are studied. However, after ranking the study participants by the responsiveness of their CD14 and THBD expression to changes of 25(OH)D3 concentrations in both tested tissues, we found in the top half of the ranked participants a significant positive correlation. From the latter 35 individuals only 3 showed a slight decrease in 25(OH)D3 concentrations, i.e. majority of them seem to benefit from the intervention irrespective of their initial serum 25(OH)D3 concentration. In fact, only 4 of the 35 participants had an initial 25(OH)D3 concentration of below 50 nM, i.e. according to the recent IoM recommendations [6] most of the participants would not have needed a vitamin D supplementation. For the other half of the study group no relationship between changes in 25(OH)D3 concentrations and VDR target gene expression could be found. These individuals showed a more individual response to vitamin D supplementation (or the lack of it) and no general conclusion could be reached from gene expression data.We suggest that analysis of the responsiveness of the genes CD14 and THBD to changes in 25(OH)D3 serum concentrations allows a categorization of the study participants. Half of the participants can be considered as conventional responders to vitamin D. These individuals have a fully functional vitamin D signaling system and their vitamin D concentrations have not reached saturation. This is proven by the down-regulation of IL6 protein in serum. IL6 has not yet been shown to be a primary VDR target, but it is known as one of the genes, via which the anti-inflammatory effect 23977191 of vitamin D is mediated [42,43]. IL6 is a marker of low-grade inflammation and has been suggested as a risk factor for type 2 diabetes [44] and cardiovascular disease [45]. Therefore, the down-regulation of IL6 protein in response to raising 25(OH)D3 serum concentrations is an indication of a beneficial effect of vitamin D3 sup.
