Alcohol disrupts TLR4 signaling in lipid rafts

描述(由申请人提供)：饮酒与感染的易感性增加和免疫反应受损有关，其机制尚不清楚。内毒素是细菌壁的主要成分，由Toll样受体(TLR)4和 辅助分子CD14，并触发各种细胞内事件，最终导致促炎细胞因子的产生。我们以前的工作表明，在单核细胞中，急性酒精通过抑制核因子B途径来损害细菌脂多糖(LPS)诱导的细胞激活，从而导致促炎细胞因子的产生减少。最近的研究表明，内毒素触发了一个大的“信号体”的形成--一个由细胞受体组成的复合体，包括TLR4、CD14、FCR、CD36、CD55、CD11b、CD18、Hsp70、Hsp90和CXCR4。信号体的形成需要坚硬的富含胆固醇的膜平台，称为膜筏。我们最近发现，通过耗尽胆固醇而破坏膜筏可以阻止内毒素诱导的细胞激活。此外，我们发现酒精可以阻止TLR4与筏子的联系。根据初步数据，我们假设酒精可能影响LP启动细胞激活的早期事件。我们推测，酒精抑制细胞激活依赖于发生在膜筏水平上的信号事件的复杂性。我们进一步假设，急性酒精的靶点是脂多糖引发的信号体重新聚集到脂筏中，从而阻止细胞激活。具体地说，我们认为酒精干扰了“信号体”的形成。这项建议的具体目的是通过探索与膜筏的共定位来确定酒精对TLR4受体复合体(TLR4、CD14、MD2、MyD88，信号体成员)A)完整性的影响；B)评估与洗涤剂耐受膜(DRM)的关联；C)检测TLR4受体复合体内蛋白质的获得/丢失；D)研究TLR4多聚体的形成。这些研究的结果应该描绘出在急性酒精暴露后导致脂多糖诱导的细胞激活受损的早期分子事件。对内毒素信号的早期步骤的研究可以确定干扰内毒素效应的策略，并设计提高对感染的免疫力的治疗方法。

DESCRIPTION (provided by applicant): Alcohol consumption is associated with increased susceptibility to infections and impaired immune responses, the mechanisms if which are not well understood. LPS, a major component of bacterial wall, is recognized by toll-like receptor (TLR) 4 and accessory molecule CD14 and triggers a variety of intracellular events that culminates in the production of pro-inflammatory cytokines. Our previous work showed that in monocytes, acute alcohol impairs bacterial lipopolysaccharide (LPS)-induced cellular activation via inhibition of NF¿B pathway to result in low pro-inflammatory cytokines production. Recent studies suggest that LPS triggers formation of a large "signalosome" - a complex of cellular receptors, including TLR4, CD14, FcR, CD36, CD55, CD11b, CD18, Hsp70, Hsp90, and CXCR4. Formation of signalosome requires rigid cholesterolrich membrane platforms, called membrane rafts. We recently identified that disruption of membrane rafts via cholesterol depletion prevents LPS-induced cell activation. Further, we identified that alcohol prevents TLR4 association with rafts. Based on preliminary data, we hypothesize that alcohol may affect the early events of LPStriggered cell activation. We postulate that inhibition cell activation by alcohol depends on the complexity of signal events that take place at the level of membrane rafts. We further hypothesized that acute alcohol targets the LPS-triggered recruitment of signalosome into lipid rafts and thus prevents cell activation. Specifically, we propose that alcohol disrupts the formation of the "signalosome". The Specific Aim of this proposal is to determine the influence of alcohol on the integrity of the TLR4 receptor complex (TLR4, CD14, MD2, MyD88, signalosome members) A) by exploring the colocalization with membrane rafts; B) evaluating the association with detergent-resistant membranes (DRMs); C) detecting acquisition/loss of proteins within the TLR4 receptor complex D) studying the formation of TLR4 multimers. Results from these studies should delineate the early molecular events that lead to impaired LPS-induced cellular activation after acute exposure to alcohol. Investigation of the early steps of LPS signaling may identify strategies for interfering with the effects of LPS and design therapeutic approaches for improving immunity against infections.