Vitamin D has been previously recognized to play important roles in human immune system and function. In the pulmonary system, vitamin D regulates the function of antimicrobial peptides, especially cathelicidin/LL-37. Human cathelicidin/LL-37 is a bactericidal, bacteriostatic, and antiviral endogenous peptide with protective immune functions. Chronic exposure to excessive alcohol has the potential to reduce levels of vitamin D (inactive vitamin D [25(OH)D3] and active vitamin D [1, 25(OH)2D3]) and leads to downregulation of cathelicidin/LL-37. Alcohol-mediated reduction of LL-37 may be partly responsible for increased incidence of more frequent and severe respiratory infections among subjects with alcohol use disorder (AUD). The objective of this study was to investigate the mechanisms by which alcohol exerts its influence on vitamin D metabolism. In addition, the aim was to establish associations between chronic alcohol exposures, levels of pulmonary vitamin D, and cathelicidin/LL-37 using broncho-alveolar lavage fluid samples of subjects with AUD and healthy controls. Findings from the experiment showed that levels of inactive vitamin D (25(OH)D3), active vitamin D (1, 25(OH)2D3), cathelicidin/LL-37, and CYP27B1 proteins were significantly reduced (P < 0.05) when compared with the matched healthy control group. However, CYP2E1 was elevated in all the samples examined. Chronic exposure to alcohol has the potential to reduce the levels of pulmonary vitamin D and results in subsequent downregulation of the antimicrobial peptide, LL-37, in the human pulmonary system.

Background Alcohol use disorders are often associated with lung disease. Alcohol exposure leads to the production of reactive oxygen species, lipid peroxidation, and formation of malondialdehyde (MDA) as well as induce the expression of cytochrome p450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA can bind to DNA forming MDA deoxyguanosine (M1dG) adducts, which have been implicated in alcohol-related cancers and cardiovascular disease. Because CYP2E1 regulates MDA production, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would modulate M1dG adduct formation and single strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue. Methods Normal human bronchial epithelial cells (HBEC) were pre-treated with 10 μM DADS for 1h, and treated with 80 mM ethanol +/− 5% cigarette smoke extract (CSE) for 3 hrs for comet assay and 6 hrs for CYP2E1, MDA, and M1dG adduct assays. C57BL/6 mice were administered 20% ethanol ad libitum in drinking water for 8 wk and exposed to whole body cigarette smoke for 5 wk. Mice were also fed a CYP2E1 inhibitor, diallyl disulfide (DADS), at 1 μM/g of feed in their daily diet for 7 wk. Whole lung tissue homogenate was used for CYP2E1, MDA, and M1dG adduct assays. Results Ethanol exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBEC attenuated this ethanol effect. Ethanol also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE +/− ethanol did not enhance these effects. In lung tissue homogenate of 8 wk alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison to control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5 wk cigarette smoke-exposed mice. Conclusions These findings suggest that CYP2E1 plays a pivotal role in alcohol-induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation.

Sickle cell disease (SCD) is a genetic disorder resulting from the presence of mutated hemoglobin S (HbS). Homozygous carriers will present with early manifestations of painful vaso-occlusive crises. SCD patients have been reported to be severely deficient in vitamin D (<20 ng/mL). Four years (2010–2014) of individual de-identified Sickle Cell Clinic of Southern Louisiana (SCCSL-SCD) patient records were analyzed for vitamin D status and the level of crisis-related ER/hospital utilization. To determine the dietary, and behavioral mediators of SCD-crisis in our study population, a cohort of 102 SCCSL-SCD patients were administered a survey that evaluated sun exposure, dietary behaviors, and pain frequency and severity. Patients with circulating levels of 25(OH)D3 less than 14.1 ng/mL reported having more crisis-related hospital visits per year (10) than patients with 25(OH)D3 serum levels >34 ng/mL. The result of the dietary survey detailed a relationship between patients who reported to have “Almost Never” consumed fish or milk in their diets and more frequent hospital stays and ER visits than those who reported consuming these products on a daily basis. Those who consumed these foods in their diet several times a month also had fewer ER visits when compared to the “Almost Never” category.

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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 (70 mM) only, diallyl disulfide (DADS) (10 μM) -only, and a co-exposure of ethanol (70 mM) and DADS (10 μM) for 10 or 24 hours. Calcidiol (50 ng/mL) and calcitriol (0.05 ng/mL) dose-response studies were conducted for 48 hours. 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)D 3 ], active vitamin D [1, 25(OH) 2 D 3 ], 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)D 3 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) 2 D 3 cellular lysate levels were lower but followed a similar trend as the 25(OH)D 3 response. LL-37 levels in primary bronchial, alveolar cells, and ThP-1 cells 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 antimicrobial peptide (LL-37) in vitro, and the co-exposure of DADS significantly attenuates ethanol-induced intracellular LL-37 depletion.