Influence of Vitamin D on Genome-Wide Expression in Natural Killer Cells

Abstract

Introduction: Vitamin D deficiency has been associated with decreased overall survival in patients with diffuse large B-cell lymphoma treated with rituximab. Natural killer cell-mediated antibody-dependent cytotoxicity is one of the main mechanisms of action of rituximab, and it has been shown to be enhanced after in vivo vitamin D supplementation. This effect was more pronounced in females. We aimed to explore molecular mechanisms behind these findings using whole transcriptome analysis of natural killer cells after vitamin D supplementation. Methods: Natural killer cells were isolated by magnetic depletion from eight otherwise healthy subjects (four females and four males) with vitamin D deficiency, before and after vitamin D supplementation to a target serum level of 65 ng/ml. RNA was extracted followed by whole transcriptome microarray analysis. Results were verified by quantitative polymerase chain reaction. For differential expression analysis, the paired t-test and two-way ANOVA were used. Comparisons were made with regard to both vitamin D status and sex. In addition, pathway analysis using gene set enrichment analysis was performed. Results: After correction for multiple testing no genes showed statistically significant differential expression in the sex-independent comparison. For hypothesis generation, genes with an unadjusted p-value < 0.01 are reported (n = 505). An impact on known vitamin D-dependent genes was demonstrated. Genes involved in cytokine signaling like IFNL3 and IL-2RB were found to be upregulated, while others like IFNG and TLR genes were downregulated. In the sex-dependent analysis 28 transcripts with statistically significant sex- and vitamin D-dependent expression after adjustment were identified, like the JPX gene which was upregulated in females. Pathway analysis highlighted the role of interferon-α genes (IFNA2, -A4, -A6, -A8, -A10) in statistically significantly upregulated pathways. Other pathways involved in the immune response were found to be significantly downregulated, mainly by downregulation of the TLR2, -5, -7 and -8 genes. Finally, the ubiquitin ligase pathway was found to be downregulated. Conclusion: Vitamin D supplementation has only a slight effect on the natural killer cell transcriptome and the small sample size used in this study limits detection of such subtle changes. Our results implicate a role for vitamin D in gene expression involving the toll-like receptor and type I/III interferon axis and its regulation through the ubiquitin ligase system. IFN-γ downregulation may well be consistent with the observation of increased lymphoma killing of natural killer cells. JPX-dependent epigenetic regulation of X-chromosome deactivation in females could explain the downregulation of genes like TLR7 and -8 which are located on the X-chromosome. It is also possible, that vitamin D exerts its effects on natural killer cells indirectly through other, regulatory cells such as dendritic cells or macrophages.