Exploratory multi-omics analysis suggests context-dependent immune responses to vitamin D₃ supplementation in PBMCs...

Vitamin D deficiency is a recognized risk factor for multiple sclerosis (MS), yet the in vivo molecular mechanisms linking vitamin D3 supplementation to immune regulation remain incompletely understood.

We performed longitudinal RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiling of peripheral blood mononuclear cells (PBMCs) from patients with relapsing-remitting MS who either initiated daily high-dose vitamin D3 supplementation or continued regular supplementation, alongside healthy individuals receiving monthly vitamin D3 bolus supplementation. Within-individual changes over the follow-up period were assessed and integrated with published THP-1-derived vitamin D receptor (VDR) binding datasets and previously validated vitamin D target genes.

PBMCs from MS patients retained stable cohort-associated transcriptional and chromatin accessibility signatures that remained distinct from those of the healthy cohort. Vitamin D3 supplementation was not associated with global transcriptomic remodeling but was associated with changes in selected immune-regulatory gene expression programs. In the daily high-dose MS group, candidate genes showing longitudinal expression changes were enriched for innate immune sensing, Toll-like receptor signaling, cytokine-mediated communication, interferon-associated responses, and immune-cell differentiation. Candidate gene sets showed limited overlap across cohorts, although a subset of immune-regulatory and established vitamin D target genes was reproducibly identified. Chromatin accessibility profiling revealed thousands of cohort-specific differentially accessible regions, whereas supplementation-associated accessibility changes were comparatively modest. Integrative analysis prioritized a small set of candidate genes supported by multiple independent datasets, including AZI2, ABCA2, and SIGLEC6.

The findings suggest selective, context-dependent candidate molecular responses to vitamin D₃ supplementation rather than global immune reprogramming. They also demonstrate the value of integrating transcriptomic and chromatin accessibility data to prioritize vitamin D-responsive pathways and genes for mechanistic validation in larger prospective and randomized studies.