Vitamin D and Lipopolysaccharide jointly induce a distinct epigenetic and transcriptional program in Human Monocytes [ATAC-seq]

Pathogen-associated molecular patterns such as lipopolysaccharide (LPS) mimic immune responses triggered by bacterial infections. The hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], supports innate immunity, but its molecular mechanisms remain incompletely understood. Here, we investigated epigenomic and transcriptomic changes in THP-1 monocytes exposed to 1,25(OH)₂D₃, LPS, or their combination over 24–48 hours. Epigenome profiling via ATAC-seq revealed that co-stimulation with 1,25(OH)₂D₃ and LPS induces substantially more chromatin accessibility changes than either treatment alone, with up to 81% of altered regions uniquely responsive to the combination. Motif enrichment analysis highlighted JUN/FOS transcription factors as key regulators of this synergistic response. Transcriptomic analysis via RNA-seq mirrored these findings, though fewer genes than chromatin regions were affected. Notably, under 1,25(OH)₂D₃-primed conditions, 333 genes exhibited synergistic expression changes upon co-treatment, meaning their responses significantly deviates from the additive effects of the individual stimulations. This includes 267 previously unrecognized as vitamin D targets. Functional annotation revealed these genes are primarily linked to monocyte and T cell differentiation, in contrast to classical vitamin D targets associated with inflammation. In conclusion, our findings uncover a distinct and cooperative regulatory program driven by vitamin D and LPS, advancing our understanding of how vitamin D shapes immune responses at the chromatin and transcriptional levels.