Evaluation of Anti-microbial Peptide (Cathelicidin/Ll-37) Levels in Ethanol-Induced Pulmonary Vitamin D Dysmetabolism

Abstract

Introduction: Vitamin D has been recognized to play an important role in human immune system and function. In the pulmonary system, vitamin D regulates the function of anti-microbial peptides such as 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 the levels of vitamin D (active and inactive) and in turn result in down-regulation of Cathelicidin/LL-37. Alcohol-mediated reduction of LL-37 maybe partly responsible for increased incidence of more frequent and severe respiratory infections among subjects with Alcohol Use Disorder (AUD).

The objective of this project was to investigate the mechanism by which alcohol exerts its influence on vitamin D metabolism and consequently (based on the observed influence) establish the relationships between chronic ethanol exposure, levels of pulmonary vitamin D and Cathelicidin/LL-37.

Materials and Methods: Human Pulmonary Alveolar/Bronchial Epithelial cell lines were grown and cultured. The cultured cells were treated with ethanol and Diallyl Disulfide (DADS). In addition, Broncho Alveolar Lavage Fluid (BALF) samples of AUD subjects and healthy controls were obtained from CoPARC (Colorado Pulmonary Alcohol Research Consortium (Denver and Atlanta Centers). Following exposure, we quantified total protein levels of vitamin D, phase one metabolizing enzymes (CYP450) and LL-37 in the cell lines and BALF. DADS has been found to have inhibitory effects on ethanol metabolic pathway mediated by CYP2E1, an enzyme that catabolize alcohol into its toxic metabolites through oxidation reaction. Study participants (AUD subjects and healthy controls) were recruited and screened using Alcohol Use Disorder Identification Test (AUDIT) score and a standardized questionnaire.

Results: The levels of 25(OH)D₃, 1,25(OH)₂D₃, Cathelicidin/LL-37, CYP27B1 proteins were significantly reduced (p < 0.05) when compared with control group (cell lines). Whereas, the level of CYP2E1 and CYP24A1 were significantly elevated following exposure to ethanol (p < 0.05), when compared to the control group. Similar trends were observed in the BALF samples when the levels of the above proteins were quantified, compared to matched healthy controls. However, upon exposure to DADS, a reversal of the above trends was observed in the quantification of respective total proteins.

Conclusion: Chronic exposure to alcohol has the potential to reduce levels of pulmonary vitamin D which in turn may result in down-regulation and/or in-activation of anti-microbial peptides, LL-37, a protein which play significant protective immune functions in the human pulmonary system.