Ethanol Changes Gene Expression of Transcription Factors and Cytokine Production of CD4+ T-Cell Subsets in PBMCs Stimulated With LPS

Background: Acute ethanol intoxication has the potential to alter immune reactivity by various pathways. The aim of this study was to investigate T-helper cell subsets transcription factors and cytokines in human peripheral blood mononuclear cells (PBMCs) following a single dose of lipopolysaccharide (LPS) with or without ethanol exposure. Methods: Human PBMCs were cultured in the presence of 100 mM ethanol and/or 100 ng/ml LPS for various time periods (1, 3, 8, and 24 hours) and analyzed for the kinetics of gene expression by quantitative real-time PCR of selected transcription factors (T-bet, GATA3, Foxp3, and ROR gamma t) and cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma). The proportion of Th17 and Treg cells was identified 24 hours after treatment with ethanol and LPS by multiparameter flow cytometry. Viability and amount of dead cells were analyzed after 24 and 48 hours by MTT assay and flow cytometry. Results: Following LPS challenge, gene expression of Foxp3 increased, whereas ROR gamma t decreased after 3 hours, GATA3 decreased within 1 hour, whereas expression of T-bet did not change at any time. Gene expression of TNF-alpha, interferon-gamma (IFN-gamma), and IL-6 peaked after 3 hours, expression of IL-10 peaked after 8 hours. Ethanol suppressed the LPS-induced gene expression of Foxp3, ROR gamma t, and T-bet after 8 hours, expression of TNF-alpha and IFN-gamma was also suppressed after 3 and 8 hours. Markers of inflammation including TNF-alpha and IL-1 beta in supernatant of PBMCs were significantly decreased, while levels of IL-10 and IL-6 remained unchanged following ethanol exposure. Furthermore, ethanol-treated cells alone or in combination with LPS had significantly fewer IL-17- and IFN-gamma-secreting CD4+ T cells but constant proportion of Treg cells when compared to control cells. Proliferation and viability of the cells were not influenced under these conditions. Conclusions: Alcohol interferes with the kinetics of Foxp3, ROR gamma t, and T-bet gene expression and the production of TNF-alpha and IL-1 ss and influences the balance of Treg/Th17 cells following LPS exposure.