Diallyl disulfide inhibits ethanol-induced pulmonary cell vitamin D and antimicrobial peptide cathelicidin depletion.

Abstract

Ethanol has been found to affect pulmonary cells by interfering with vitamin D metabolism and pulmonary defense mechanisms. The objective of this study was to understand the mechanisms of ethanol's disruptive influence on the vitamin D pathway and inhibition of anti-microbial peptide cathelicidin (LL-37). Bronchial epithelial cells (BEAS-2Bs), primary human bronchial epithelial cells (HBECs), primary human alveolar epithelial cells (HPAEpiCs), and human monocyte cells (THP-1s) were used in this study. These cells were cultured and exposed to different treatment groups: medium-only control, ethanol (70mM) only, diallyl disulfide (DADS) (10μM) -only, and a co-exposure of ethanol (70mM) and DADS (10μM) for 10 or 24h. Calcidiol (50ng/mL) and calcitriol (0.05ng/mL) dose-response studies were conducted for 48h. After incubation, cells were trypsinized, lysed, and centrifuged, and the cellular lysate was prepared for assay. Protein was quantified, and levels of inactive vitamin D [25(OH)D3], active vitamin D [1, 25(OH)2 D3], and anti-microbial peptides (cathelicidin/LL-37) in the samples were assayed using commercially available ELISA kits. In the ethanol-exposed group, cellular lysate concentrations of 25(OH)D3 and LL-37 were significantly reduced by 30%, and 40% in BEAS-2B cells, and 35% and 80% in HPAEpi cells respectively. Overall 1, 25(OH)2D3 cellular lysate levels were lower but followed a similar trend as the 25(OH)D3 response. LL-37 levels in primary bronchial, alveolar cells, and ThP-1cells were statistically reduced in ethanol-exposed groups (60%, 80%, and 65%, respectively) when compared with control. Following the addition of DADS, levels of LL-37 were recovered to within control levels for all three cell types. This study establishes two clinically relevant observations: that the exposure of pulmonary epithelial and monocyte cells to physiologically relevant levels of excessive ethanol selectively disrupts the activation of pulmonary vitamin D and inhibits the presence of anti-microbial peptide (LL-37) invitro, and the co-exposure of DADS significantly attenuates ethanol-induced intracellular LL-37 depletion.