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

描述(申请人提供)：酒精(乙醇，乙醇)对人体有多种影响，从对心血管系统的有益影响到对其他系统的有害影响，包括对免疫系统的抑制。乙醇的这种多功能作用的机制在很大程度上是未知的。本研究的目的是明确乙醇对钙依赖的内毒素触发的巨噬细胞(MF)激活调控的分子机制的影响。我们的初步结果表明，乙醇抑制病原体介导的MF激活，特别是在急性乙醇暴露时，MF产生的促炎细胞因子较少。当细菌衍生产物时，脂多糖(LPS)通过Toll样受体4(TLR4)触发细胞激活。我们提供了新的初步数据，即TLR4与脂多糖的结合触发了MF胞浆钙的快速增加。此外，钙耗竭或化学阻断钙内流可阻断内毒素诱导的肿瘤坏死因子？在明尼苏达州的生产。乙醇抑制脂多糖诱导的CaMKII的磷酸化，CaMKII是一种钙依赖的激酶，在初始TLR4信号复合体的组装中起关键作用。乙醇刺激调节肿瘤坏死因子？的核因子--核因子-AT的钙依赖性激活。制作。根据我们的初步数据，我们推测，钙在TLR4介导的MF激活中起关键作用，其次，乙醇诱导的钙介导的信号在MF中的损伤。我们假设a)乙醇条件下胞浆内钙的调节通过初始的TLR4信号复合体导致信号缺陷，和/或b)酒精调节先前未知/低估的内毒素触发的钙-钙调蛋白-钙调神经磷酸酶-CaMKII-核因子-AT通路。我们推测，急性乙醇条件性损伤内毒素诱导的MF激活的机制包括改变钙稳态和诱导大电导钙激活钾通道的调制。这项建议解决了我们的假设，揭示了钙在乙醇诱导的TLR4介导的天然免疫激活损伤中的作用。与公共健康相关：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，包括抑制免疫系统。乙醇的这种多功能作用的机制在很大程度上是未知的。这项研究计划的总体目标是确定酒精对巨噬细胞激活调控的分子机制的影响。巨噬细胞产生促炎细胞因子--肿瘤坏死因子？当细菌衍生的产物，脂多糖(LPS)与Toll样受体4(TLR4)结合。酒精抑制脂多糖诱导的肿瘤坏死因子？制作。我们提供了新的初步数据，即TLR4的参与增加了MF的胞浆钙离子。更重要的是，与急性酒精类似，钙耗竭或化学阻断可消除内毒素诱导的肿瘤坏死因子？制作。我们推测，第一，钙在TLR4介导的MF激活中起关键作用；第二，酒精调节MF中的钙稳态。我们假设a)酒精对钙的调节导致初始TLR4信号复合体的缺陷形成，和/或b)酒精调节先前未知/低估的脂多糖触发的钙-钙调素-钙调神经磷酸酶-钙调素-钙调素-NFAT通路。我们进一步假设急性酒精对钙依赖的大电导钙激活的钾通道有调节作用。这一建议将解决我们的假设，以揭示钙在乙醇诱导的TLR4介导的天然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.