Inhibition of Methicillin-resistant Staphylococcus aureus-induced cytokines mRNA production in human bone marrow derived mesenchymal stem cells by 1,25-dihydroxyvitamin D3

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)(2)D-3) has potent immunoregulatory properties, but the mechanism remains poorly understood. The genomic action of 1,25(OH)(2)D-3 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)(2)D-3 inhibits MRSA-induced cytokines synthesis in hMSCs via inhibition of NF-kappa B transcription factor. Finally, we explored the regulatory role of 1,25(OH)(2)D-3 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, TNF alpha 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)(2)D-3 followed by MRSA co culture inhibited nuclear translocation of NF kappa B p65, reduced expression of NR4A2 and pro-inflammatory cytokines IL-8, IL-6, and TNF alpha in hMSCs. Further, NF-kappa B-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)(2)D-3 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)(2)D-3-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)(2)D-3-VDR may exhibits its anti-inflammatory properties in MRSA-stimulated infection by inhibiting nuclear translocation of NF-kappa B-p65 and transcripts of IL-8, IL-6, TNF alpha, and NR4A2 in hMSCs. Finally, 1,25(OH)(2)D-3-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.