Transcriptomic profiling of immune modulation induced by vitamin D3 in the VitDPAS and VitDHiD cohort studies

Abstract

The VitDPAS study (NCT06104111) was designed as a medical experiment to assess the in vivo effects of vitamin D on immune responses. This study enrolled 45 healthy individuals from Olsztyn, Poland, who received a body weight-adjusted bolus dose of vitamin D₃ (1,000 IU/kg). Transcriptome-wide differential gene expression analysis of peripheral blood mononuclear cells, collected before and 24 h after supplementation, identified 758 significantly responsive genes (p < 0.05). By correlating individual gene expression changes with alterations in vitamin D status, participants were categorized into three response groups: 17 high responders, 19 mid responders, and 9 low responders. A comparative analysis with the VitDHiD study (NCT03537027), conducted on a Finnish cohort of 25 healthy participants, revealed 232 overlapping target genes, enabling an integrated assessment of vitamin D responsiveness across all 70 individuals. Applying a more stringent statistical threshold (false discovery rate < 0.05) highlighted 26 shared target genes, demonstrating a consistent in vivo response to vitamin D₃ across both cohorts. The modulation of inflammatory processes, mediated primarily via tumor necrosis factor and nuclear factor κB signaling pathways, emerged as a shared effect, highlightening the immunomodulatory potential of vitamin D as a key function of the vitamin in healthy individuals.

Keywords: Inflammatory response; Transcriptome; Vitamin D; Vitamin D intervention studies; Vitamin D response index; Vitamin D target genes.

Fig. 1

Transcriptome-wide analysis of vitamin D₃ bolus supplementation in the VitDPAS cohort. (A) The quality of transcriptome datasets (of 758 target genes) from the 45 VitDPAS participants at d0 and d1 was assessed using dimensionality reduction techniques, visualized through a MDS plot. (B) A Volcano plot was employed to illustrate the impact of vitamin D₃ supplementation on the expression of 758 significantly regulated genes (p < 0.05). Selected target genes are highlighted.

Fig. 2

Classification of VitDPAS study participants. (A) The 45 VitDPAS participants were classified into high (green), mid (yellow), and low (red) responders based on the expression profiles of all 758 in vivo vitamin D target genes. (B) Comparison of classification methods utilizing various subsets of vitamin D target genes to evaluate the robustness of the segregation approach.

Fig. 3

Overlap and classification of vitamin D target genes between VitDPAS and VitDHiD studies. (A) A Venn diagram highlights the intersection of 758 significant (p < 0.05) in vivo vitamin D target genes identified in the VitDPAS study and 1,654 target genes from the VitDHiD study. (B) The quality of transcriptome datasets for the 232 shared target genes at d0 and d1 was assessed using dimensionality reduction, depicted in a MDS plot. (C) Based on the expression profiles of the 232 common target genes, the 70 participants across both studies were categorized into high (green), mid (yellow), and low (red) vitamin D responders.

Fig. 4

Overlap and functional classification of vitamin D target genes between VitDPAS and VitDHiD studies. (A) A Venn diagram illustrates the overlap between 70 highly significant (FDR < 0.05) in vivo vitamin D target genes identified in the VitDPAS study and 452 target genes from the VitDHiD study. (B) The 26 shared vitamin D target genes are grouped into distinct functional categories. Gene interactions, derived from the STRING database (Supplementary Figure S8 online), are depicted with connecting lines. Upregulated genes are shown in green, while downregulated genes are represented in red.