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

描述（由申请人提供）：酒精（乙醇，EtOH）消耗对人体表现出各种各样的影响，从对心血管系统的有益影响到对其他系统的有害影响，包括免疫系统的抑制。导致EtOH这种多功能作用的机制在很大程度上是未知的。该提案的目的是确定EtOH对参与调节Ca 2+依赖性LPS触发的巨噬细胞（Mf）激活的分子机制的影响。我们的初步结果表明，乙醇抑制病原体介导的MF激活，特别是，急性乙醇暴露MF产生较少的促炎细胞因子TNF？当细菌衍生产物时，脂多糖（LPS）通过Toll样受体4（TLR 4）触发细胞活化。我们提供了新的初步数据，参与TLR 4与LPS触发细胞质Ca 2+在Mf的快速增加。此外，Ca 2+耗竭或化学阻断Ca 2+内流取消LPS诱导的TNF？生产MF。EtOH损害LPS诱导的CaMKII磷酸化，CaMKII是一种Ca 2+依赖性激酶，在初始TLR 4信号复合物的组装中起关键作用。EtOH刺激Ca 2+依赖性激活NF-AT，一种调节TNF？生产基于我们的初步数据，我们推测，Ca 2+在TLR 4介导的Mf激活和第二，EtOH诱导的Ca 2+介导的Mf信号转导的损伤中起着关键作用。我们假设a）细胞质中Ca 2+的EtOH条件调节导致通过初始TLR 4信号传导复合物的缺陷信号传导，和/或B）酒精调节先前未知/低估的LPS触发的Ca 2 +-钙调蛋白-钙调磷酸酶-CaMK II-NF-AT途径。我们推测，急性EtOH条件损害LPS诱导的Mf激活的机制包括改变Ca 2+稳态和诱导大电导Ca 2+激活钾通道的调制。这一建议解决了我们的假设，以解开EtOH诱导的TLR 4介导的先天免疫激活的损害中的作用，钙。公共卫生相关性：酒精消费对人体表现出各种各样的影响，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，包括抑制免疫系统。导致EtOH这种多功能作用的机制在很大程度上是未知的。这项研究的总体目标是确定酒精对参与调节巨噬细胞活化的分子机制的影响。巨噬细胞（Mf）产生促炎细胞因子TNF？当细菌衍生的产物时，脂多糖（LPS）与Toll样受体4（TLR 4）结合。酒精抑制LPS诱导的TNF？生产我们提供了新的初步数据，TLR 4的参与增加了Mf中的细胞质Ca 2+。更重要的是，类似于急性酒精，Ca 2+耗竭或化学阻断废除LPS诱导的TNF？生产在这里，我们假设，第一，Ca 2+在TLR 4介导的Mf激活中起着关键作用，第二，酒精调节Mf中的Ca 2+稳态。我们假设a）酒精条件性调节Ca 2+导致初始TLR 4信号复合物的缺陷形成，和/或B）酒精调节先前未知/低估的LPS触发的Ca 2 +-钙调蛋白-钙调磷酸酶-CaMKII-NFAT途径。我们进一步假设，急性酒精调节钙依赖性大电导钙激活钾通道。该建议将解决我们的假设，以解开EtOH诱导的TLR 4介导的先天免疫激活的损害中的作用的Ca 2+。

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.