Alcohol disrupts TLR4 signaling in lipid rafts

描述（由申请人提供）：饮酒与感染易感性增加和免疫反应受损有关，其机制尚不清楚。LPS是细菌壁的主要成分，被Toll样受体（TLR）4识别， 辅助分子CD 14并触发多种细胞内事件，最终导致促炎细胞因子的产生。我们之前的工作表明，在单核细胞中，急性酒精通过抑制NF ² B途径损害细菌脂多糖（LPS）诱导的细胞活化，导致促炎细胞因子产生较低。最近的研究表明，LPS触发形成一个大的“信号体”-细胞受体的复合物，包括TLR 4，CD 14，FcR，CD 36，CD 55，CD 11b，CD 18，Hsp 70，Hsp 90和CXCR 4。信号体的形成需要刚性的富含胆固醇的膜平台，称为膜筏。我们最近发现，通过胆固醇耗竭破坏膜筏可以阻止LPS诱导的细胞活化。 此外，我们发现酒精可以阻止TLR 4与筏的结合。基于初步数据，我们假设酒精可能影响LPS触发的细胞活化的早期事件。我们假设酒精对细胞活化的抑制作用依赖于膜筏水平上发生的信号事件的复杂性。我们进一步假设急性酒精靶向LPS触发的信号体招募到脂筏中，从而阻止细胞活化。具体来说，我们认为酒精破坏了“信号体”的形成。本提案的具体目的是确定酒精对TLR 4受体复合物（TLR 4、CD 14、MD 2、MyD 88、信号体成员）完整性的影响：A）通过探索与膜筏的共定位; B）评价与耐洗涤剂膜（DRM）的相关性; C）检测TLR 4受体复合物内蛋白质的获得/损失D）研究TLR 4多聚体的形成。这些研究的结果应该描述急性酒精暴露后导致LPS诱导的细胞活化受损的早期分子事件。对LPS信号传导的早期步骤的研究可以确定干扰LPS作用的策略，并设计用于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.