Department of Anaesthesiology and Intensive Care Medicine, Campus Charité Mitte und Charité Campus Virchow-Klinikum, Charité-Universitätsmedizin, Berlin, Germany.
Alcohol Clin Exp Res. 2011 Apr;35(4):621-31. doi: 10.1111/j.1530-0277.2010.01376.x. Epub 2010 Dec 8.
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.
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 RORγt) and cytokines (TNF-α, IL-6, IL-10, and IFN-γ). 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.
Following LPS challenge, gene expression of Foxp3 increased, whereas RORγt decreased after 3 hours, GATA3 decreased within 1 hour, whereas expression of T-bet did not change at any time. Gene expression of TNF-α, interferon-γ (IFN-γ), and IL-6 peaked after 3 hours, expression of IL-10 peaked after 8 hours. Ethanol suppressed the LPS-induced gene expression of Foxp3, RORγt, and T-bet after 8 hours, expression of TNF-α and IFN-γ was also suppressed after 3 and 8 hours. Markers of inflammation including TNF-α and IL-1β 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-γ-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.
Alcohol interferes with the kinetics of Foxp3, RORγt, and T-bet gene expression and the production of TNF-α and IL-1ß and influences the balance of Treg/Th17 cells following LPS exposure.
急性乙醇中毒可通过多种途径改变免疫反应。本研究旨在探讨人外周血单个核细胞（PBMC）在单次脂多糖（LPS）暴露或不暴露乙醇的情况下，T 辅助细胞亚群转录因子和细胞因子的变化。
将人 PBMC 在 100mM 乙醇和/或 100ng/ml LPS 存在的情况下培养不同时间（1、3、8 和 24 小时），通过定量实时 PCR 分析选定转录因子（T-bet、GATA3、Foxp3 和 RORγt）和细胞因子（TNF-α、IL-6、IL-10 和 IFN-γ）的基因表达动力学。用乙醇和 LPS 处理 24 小时后，通过多参数流式细胞术鉴定 Th17 和 Treg 细胞的比例。用 MTT 测定法和流式细胞术分析 24 小时和 48 小时后细胞活力和死亡细胞数量。
LPS 刺激后，Foxp3 的基因表达在 3 小时后增加，而 RORγt 在 3 小时后减少，GATA3 在 1 小时内减少，而 T-bet 的表达在任何时间都没有改变。TNF-α、干扰素-γ（IFN-γ）和 IL-6 的基因表达在 3 小时后达到峰值，IL-10 的基因表达在 8 小时后达到峰值。乙醇在 8 小时后抑制 LPS 诱导的 Foxp3、RORγt 和 T-bet 的基因表达，在 3 和 8 小时后也抑制 TNF-α和 IFN-γ的基因表达。乙醇暴露后，PBMC 上清液中包括 TNF-α和 IL-1β在内的炎症标志物显著减少，而 IL-10 和 IL-6 的水平保持不变。此外，与对照细胞相比，单独用乙醇处理或与 LPS 联合处理的细胞产生的 IL-17 和 IFN-γ分泌的 CD4+T 细胞明显减少，但 Treg 细胞的比例保持不变。在这些条件下，细胞的增殖和活力不受影响。
酒精干扰 LPS 暴露后 Foxp3、RORγt 和 T-bet 基因表达的动力学以及 TNF-α 和 IL-1β的产生，并影响 Treg/Th17 细胞的平衡。
