Acute ethanol exposure impairs macrophage function

急性乙醇暴露损害巨噬细胞功能

• 批准号: 7331051
• 负责人: John Karavitis
• 金额: $ 2.63万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7331051
• 关键词: Abbreviations; Actins; Acute; Alcohol consumption; Alcohols; Alveolar Macrophages; Antigen-Presenting Cells; Attenuated; Binding; Blood; Bronchoalveolar Lavage; Burn injury; CR-3; Cause of Death; Chronic; Clinical; Cytolysis; Cytoskeleton; Data; Detection; Development; Equilibrium; Ethanol; Exhibits; Extracellular Signal Regulated Kinases; Focal Adhesions; GTPase-Activating Proteins; Host Defense; Hour; Immunity; Infection; Injection of therapeutic agent; Injury; Interleukin-10; Interleukin-6; Lead; Left; Ligand Binding; Ligands; Lipopolysaccharides; MAP Kinase Gene; MAPK14 gene; Macrophage-1 Antigen; Measurement; Measures; Mediating; Microscopic; Microscopy; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Nitric Oxide; Numbers; Opportunistic Infections; Pathway interactions; Patients; Pattern; Phagocytosis; Phagosomes; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Pneumonia; Prevalence; Production; Property; Proteins; Pseudomonas aeruginosa; Publishing; Rate; Receptor Aggregation; Receptor Signaling; Respiratory Tract Infections; Saline; Signal Transduction; Site; Surface; T-Lymphocyte; Techniques; Testing; Toll-Like Receptor 2; Toll-like receptors; Trauma; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Western Blotting; Work; alcohol exposure; base; chronic alcohol ingestion; cytokine; enhanced green fluorescent protein; human NOS2A protein; human TNF protein; immune function; immunocytochemistry; improved; in vivo; injured; intraperitoneal; macrophage; monocyte; particle; pathogen; receptor; receptor expression; rho; statistics; therapeutic target; time use; toll-like receptor 4

DESCRIPTION (provided by applicant): Both acute and chronic alcohol consumption has been shown to exacerbate the complications exibited after trauma or burn injury. Alcohol also has been shown to increase the prevalence of pneumonia and other opportunistic infections which normally would be controlled. We propose to elucidate the mechanisms that cause macrophages to exhibit phagocytosis after ethanol exposure. Our model will utilize the murine acute ethanol mode that our lab has previously worked with. We predict that alveolar macrophages isolated from mice that have been treated with ethanol will exhibit a decrease phagocytosis independent on the type of receptors mediated but the pathway of inhibition may be different. Our first aim will focus on testing the degree of decreased ex vivo phagocytosis in the in vivo ethanol treated macrophages; focusing on targeting the TLR-4, TLR-2, Fcg-R, and CR-3 mediated pathways. We will utilize microscopic techniques to access the degree of inhibition. The second aim is to determine whether acute ethanol exposure effects binding and internalization of a pathogen by alveolar macrophages. The first part of this aim will focus on studying the ability of these macrophages to bind to beads coated with specific ligands to TLR-2, FcgR, and CR3. This will be followed by measuring receptor expression on the surface of the macrophage as well as receptor aggregation of the receptor to the coated bead. These data will reveal if decreases of specific ligand binding in ethanol exposed macrophages is due alterations in receptor expression or the inability of the receptor to cluster at the site of the phagolytic particle. The second part of this aim will focus on measuring the rate and degree of focal adhesion protein recruitment to the site of the forming phagosome. We will use time lapse microscopy and immunocytochemistry to measure these levels. Aim 3 of our study will focus on the downstream signaling, specifically the balance of Rac and Rho activation, as well as the production capability of alveolar macrophages to produce nitric oxide, a molecule involved in the lysis of ingested pathogens. We will utilize western blotting techniques to measure Rho-GTP, Rac-GTP, and total Rho and Rac, and a nitric oxide detection kit for nitric oxide measurements. The studies proposed in this application are necessary to uncover how ethanol exposure modulates the ability of alveolar macrophage to function properly. Understanding how ethanol decreases phagocytosis can lead us to the development of therapeutic targets that can potentially help in the clearance of a pathogen in patients who have consumed alcohol.

描述（由申请人提供）：急性和慢性酒精消耗均已证明会加剧创伤或烧伤后体现的并发症。还显示酒精会增加肺炎和其他机会感染的患病率，这些感染通常会受到控制。我们建议阐明导致巨噬细胞在乙醇暴露后表现出吞噬作用的机制。我们的模型将利用我们实验室以前与之合作的鼠急性乙醇模式。我们预测，从已接受乙醇治疗的小鼠中分离出的肺泡巨噬细胞将表现出降低的吞噬作用，而对介导的受体类型的类型无关，但抑制途径可能不同。我们的第一个目标将集中于测试体内治疗的巨噬细胞中体内吞噬作用降低的程度。专注于靶向TLR-4，TLR-2，FCG-R和CR-3介导的途径。我们将利用微观技术来获得抑制程度。第二个目的是确定急性乙醇暴露是否影响肺泡巨噬细胞对病原体的结合和内在化。该目标的第一部分将集中于研究这些巨噬细胞与特定配体覆盖的珠子与TLR-2，FCGR和CR3结合的能力。之后，将测量巨噬细胞表面的受体表达以及受体对涂层珠的受体聚集。这些数据将揭示乙醇暴露巨噬细胞中特定配体结合的降低是应有的受体表达的改变还是受体无法在吞噬颗粒部位聚类的受体。该目标的第二部分将集中于测量对形成吞噬体部位的局灶性粘附蛋白募集的速率和程度。我们将使用延时显微镜和免疫细胞化学来测量这些水平。我们研究的目标3将集中于下游信号传导，特别是RAC和RHO激活的平衡，以及肺泡巨噬细胞产生一氧化氮的生产能力，这是参与摄入的病原体裂解的分子。我们将利用蛋白质印迹技术来测量Rho-GTP，RAC-GTP和总Rho和RAC，以及一氧化氮检测试剂盒进行一氧化氮测量。 在本应用中提出的研究对于发现乙醇暴露如何调节肺泡巨噬细胞正常运行的能力是必要的。了解乙醇如何减少吞噬作用会导致我们发展治疗靶标，这些靶标有可能有助于清除饮酒患者的病原体。