BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is the predominant cause of bone infection. Toll like receptors (TLRs) are an important segments of host response to infection and are expressed by a variety of cells including human mesenchymal stem cells (hMSCs). The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunoregulatory properties, but the mechanism remains poorly understood. The genomic action of 1,25(OH)2D3 is mediated by vitamin D receptor (VDR), hormone-regulated transcription factor. VDR interacts with co-activators and co-repressors are associated with chromatin histone modifications and transcriptional regulation. The aim of our study is to explore MRSA-induced TLRs-mediated pro-inflammatory cytokines expression in hMSCs. Further, we hypothesized that 1,25(OH)2D3 inhibits MRSA-induced cytokines synthesis in hMSCs via inhibition of NF-кB transcription factor. Finally, we explored the regulatory role of 1,25(OH)2D3 in MRSA-mediated global epigenetic histone H3 mark, such as, trimethylated histone H3 lysine 9 (H3K9me3), which is linked to gene silencing. RESULTS: Quantitative PCR data revealed that MRSA-infection predominantly induced expression of TLRs 1, 2, 6, NR4A2, and inflammatory cytokines IL-8, IL-6, TNFα in hMSCs. MRSA-mediated TLR ligands reduced osteoblast differentiation and increased hMSCs proliferation, indicating the disrupted multipotency function of hMSCs. Pretreatment of 1,25(OH)2D3 followed by MRSA co-culture inhibited nuclear translocation of NF-кB-p65, reduced expression of NR4A2 and pro-inflammatory cytokines IL-8, IL-6, and TNFα in hMSCs. Further, NF-κB-p65, VDR, and NR4A2 were present in the same nuclear protein complex, indicating that VDR is an active part of the nuclear protein complexes for transcriptional regulation. Finally, 1,25(OH)2D3 activated VDR, restores the global level of H3K9me3, to repress MRSA-stimulated inflammatory cytokine IL-8 expression. Pretreatment of 5-dAZA, DNA methylatransferases (Dnmts) inhibitor, dramatically re-expresses 1,25(OH)2D3-MRSA-mediated silenced IL-8 gene. CONCLUSIONS: This data indicates that TLR 1, 2, and 6 can be used as markers for localized S. aureus bone infection. 1,25(OH)2D3-VDR may exhibits its anti-inflammatory properties in MRSA-stimulated infection by inhibiting nuclear translocation of NF-kB-p65 and transcripts of IL-8, IL-6, TNFα, and NR4A2 in hMSCs. Finally, 1,25(OH)2D3-activated VDR, acting as an epigenetic regulator, inhibits synthesis of cytokines in MRSA-stimulated infection by restoring the global level of H3K9me3, a histone H3 mark for gene silencing.