Acute alcohol and calcium-dependent LPS-triggered activation of macrophages

描述（由申请人提供）：酒精（乙醇，乙醚）消费对人体有各种各样的影响，从对心血管系统的有益影响到对其他系统的有害影响，包括抑制免疫系统。导致EtOH这种多用途效应的机制在很大程度上是未知的。本提案的目的是确定EtOH对Ca2+依赖性lps触发的巨噬细胞（Mf）激活调控的分子机制的影响。我们的初步结果表明，EtOH抑制病原体介导的Mf激活，特别是在急性EtOH暴露后，Mf产生的促炎细胞因子TNF？当细菌衍生的产物脂多糖（LPS）通过toll样受体4 （TLR4）触发细胞活化。我们提供了新的初步数据，表明TLR4与LPS的结合会触发Mf细胞质Ca2+的快速增加。此外，Ca2+耗尽或Ca2+内流的化学阻断可消除lps诱导的TNF？产量在Mf。EtOH损害了lps诱导的CaMKII的磷酸化，CaMKII是一种Ca2+依赖性激酶，在初始TLR4信号复合物的组装中起关键作用。EtOH刺激Ca2+依赖性NF-AT的激活，NF-AT是一种调节TNF？生产。根据我们的初步数据，我们假设Ca2+在tlr4介导的Mf激活中起关键作用，其次，etoh诱导的Mf中Ca2+介导的信号通路损伤。我们假设a) EtOH条件下细胞质中Ca2+的调节通过初始TLR4信号复合物导致信号缺陷，和/或b)酒精调节以前未知/低估的lps触发的Ca2+-钙调素-钙调蛋白- camkii - nf - at途径。我们假设lps诱导的急性etoh条件损伤的机制包括Ca2+稳态的改变和诱导大电导Ca2+激活钾通道的调节。这一建议解决了我们的假设，揭示Ca2+在etoh诱导的tlr4介导的先天免疫激活损伤中的作用。公共卫生相关性：饮酒对人体有多种影响，从对心血管系统的有益影响到对所有其他系统的有害影响，包括免疫系统的抑制。导致EtOH这种多用途效应的机制在很大程度上是未知的。本研究计划的总体目标是确定酒精对巨噬细胞活化调节的分子机制的影响。巨噬细胞（Mf）产生促炎细胞因子TNF？当细菌衍生的产物脂多糖（LPS）与toll样受体4 （TLR4）结合时。酒精抑制lps诱导的TNF？生产。我们提供了新的初步数据，表明TLR4的参与增加了Mf的细胞质Ca2+。更重要的是，与急性酒精相似，Ca2+耗尽或化学阻断可消除lps诱导的TNF？生产。首先，我们假设；Ca2+在tlr4介导的Mf激活中起关键作用，其次，酒精调节Mf中的Ca2+稳态。我们假设a)酒精条件下Ca2+的调节导致初始TLR4信号复合物的形成缺陷，和/或b)酒精调节以前未知/低估的lps触发的Ca2+-钙调素-钙调蛋白- camkii - nfat途径。我们进一步假设急性酒精调节Ca2+依赖的大电导Ca2+激活钾通道。该建议将解决我们的假设，以揭示Ca2+在etoh诱导的tlr4介导的先天免疫激活损伤中的作用。

DESCRIPTION (provided by applicant): Alcohol (ethanol, EtOH) consumption exhibits a wide variety of effect on the human body, ranging from beneficial effect on the cardiovascular system to detrimental effects on other systems, including suppression of the immune system. The mechanisms leading to such versatile effect of EtOH are largely unknown. The goal of this proposal is to define the influence of EtOH on molecular mechanisms involved in regulation of Ca2+-dependent LPS-triggered macrophage (Mf) activation. Our preliminary results suggest that EtOH inhibits pathogen-mediated Mf activation, specifically, upon acute EtOH exposure Mf produce less pro-inflammatory cytokine TNF? when a bacterial-derived product, lipopolysaccharide (LPS) triggers cell activation via Toll-like receptor 4 (TLR4). We provide novel preliminary data that engagement of TLR4 with LPS triggers rapid increase in cytoplasmic Ca2+ in Mf. Moreover, Ca2+ depletion or chemical block of Ca2+ influx abolished LPS-induced TNF? production in Mf. EtOH impairs LPS-induced phosphorylation of CaMKII, a Ca2+-dependent kinase with key role in assembly of initial TLR4 signaling complex. EtOH stimulates Ca2+-dependent activation of NF-AT, a nuclear factor that regulates TNF? production. Based on our preliminary data, we postulated that Ca2+ plays a key role in TLR4-mediated Mf activation and second, EtOH-induced impairment of Ca2+-mediated signaling in Mf. We hypothesize that a) EtOH- conditioned modulation of Ca2+ in the cytoplasm leads to defective signaling via initial TLR4 signaling complex, and/or b) alcohol modulates the previously unknown/under-estimated LPS-triggered Ca2+-calmodulin-calcineurin-CaMKII-NF-AT pathway. We hypothesized that the mechanisms of the acute EtOH-conditioned impairment of LPS-induced Mf activation include alterations of Ca2+ homeostasis and induce modulation of large conductance Ca2+ activated potassium channels. This proposal addresses our hypothesis to unravel the role of Ca2+ in EtOH-induced impairment of TLR4-mediated innate immune activation. PUBLIC HEALTH RELEVANCE: Alcohol consumption exhibits a wide variety of effect on human body, ranging from beneficial effect on cardiovascular system to detrimental effects on all other system, including suppression of the immune system. The mechanisms leading to such versatile effect of EtOH are largely unknown. The overall goal of this research proposal is to define the influence of alcohol on the molecular mechanisms involved in regulation macrophage activation. Macrophages (Mf) produce pro-inflammatory cytokine TNF? when a bacterial- derived product, lipopolysaccharide (LPS) binds to the Toll-like receptor 4 (TLR4). Alcohol inhibits LPS-induced TNF? production. We provide novel preliminary data that engagement of TLR4 increases the cytoplasmic Ca2+ in Mf. More importantly and similar to acute alcohol, Ca2+ depletion or chemical block abolished the LPS-induced TNF? production. Here we postulated that, first; Ca2+ plays a key role in TLR4-mediated Mf activation and second, alcohol modulates the Ca2+ homeostasis in Mf. We hypothesize that a) alcohol-conditioned modulation of Ca2+ leads to defective formation of the initial TLR4 signaling complex, and/or b) alcohol modulates the previously unknown/under-estimated LPS-triggered Ca2+-calmodulin-calcineurin-CaMKII-NFAT pathway. We further hypothesized that acute alcohol modulates the Ca2+-dependent large conductance Ca2+ activated potassium channels. This proposal will address our hypothesis in order to unravel the role of Ca2+ in EtOH-induced impairment of TLR4-mediated innate immune activation.