Vitamin D-Mediated Defense Mechanisms Against KSHV in Oral Epithelial Cells

Objectives: KSHV is shed via saliva and its infection of oral epithelial cells is important in both the course of disease and the spread of the virus. “Healthy” serum levels of vitamin D₃ have been associated with decreased viral infections and controlled KSHV-associated tumors in mouse models. The goal of this study is to explore the role of vitamin D in antiviral defense of oral epithelial cells against KSHV.
Methods: Immortalized human oral keratinocytes (OKF6/Tert-1) were treated with 10 nM 1,25-dihydroxyvitamin D₃ (1,25D₃) or ethanol (EtOH) for 24 hours and antimicrobial peptide LL-37 release was quantified via ELISA. LL-37 inactivation of KSHV-BAC16 was measured via co-incubation for one hour before adding to HEK293 cells and counting GFP-positive cells. OKF6/Tert-1 cells were also treated with 5 μM viral mimetic unmethylated CpG DNA (CpG) along with 10 nM 1,25D₃ or EtOH. RT-qPCR was performed to measure mRNA expression after six hours and IRF3 translocation to the nucleus was measured via densitometric analysis of immunofluorescence after two hours.
Results: 1,25D₃ treatment led to an increase in the media:intracellular ratio of LL-37 concentration compared to EtOH (1.2 and 0.5). Co-incubation of KSHV with 20 μg/ml LL-37 led to a two-fold decrease in KSHV infection. Co-treatment with CpG and 1,25D₃ exhibited lower Type I interferon expression compared to CpG and EtOH (1.89 and 9.30, respectively, for IFNA4 and 0.99 and 2.08 for IFNB1). CpG and 1,25D₃ treatment exhibited statistically comparable IRF3 cytoplasmic localization as untreated cells (~20%), while the localization for CpG and EtOH co-treatment was significantly higher (~30%).
Conclusions: Induction of LL-37 can inhibit initial infection, and modulation of IRF3 translocation, a protein targeted by KSHV, can preserve downstream interferon signaling that protects infected cells from viral spread. Vitamin D holds promise as a potential way to decrease KSHV infection of oral epithelial cells.