Identifying the molecular target for macrophage activation by chlorpyrifos

确定毒死蜱激活巨噬细胞的分子靶标

• 批准号: 10467360
• 负责人: Michael S Cohen
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-25 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10467360
• 关键词: Acetylcholine; Animals; Apoptosis; Asthma; Binding; Binding Proteins; Brain; Bronchoconstriction; CRISPR/Cas technology; Cavia; Cells; Chemistry; Chlorpyrifos; Cholinesterase Inhibitors; Cholinesterases; Chronic; Data; Development; Diazinon; Dichloromethylene Diphosphonate; Digestion; Dose; Electric Stimulation; Encapsulated; Environmental Pollution; Etanercept; Exposure to; Frequencies; Genes; Health; Human; In Vitro; Insecta; Ligands; Liposomes; Lung; Macrophage Activation; Measures; Mediating; Messenger RNA; Methods; Microglia; Molecular Target; Muscarinic M2 Receptor; Nerve; Neuraxis; Organophosphates; Parathion; Parents; Pesticides; Physiological; Prevention approach; Proteins; Reflex action; Risk; Risk Factors; Role; Signal Transduction; Testing; Time; Tissues; airway hyperresponsiveness; cholinesterase 2; experimental study; genome-wide; macrophage; new therapeutic target; pesticide exposure; prevent; response; toxic organophosphate insecticide exposure; vagus nerve stimulation

Chronic exposure to low levels of organophosphate pesticides is associated with increased risk for asthma. We have shown in experimental animals that low-level exposure activates lung macrophages to produce TNFa, which in turn decreases expression and function of inhibitory M2 muscarinic receptors on airway parasympathetic nerves, increasing the release of acetylcholine and potentiating reflex bronchoconstriction. We hypothesize that molecular targets responsible for activation of macrophages by organophosphates can be identified. To do so, we propose two specific aims: Specific Aim #1: Use photocrosslinking and click chemistry to identify potential targets that chlorpyrifos binds to on macrophages. We will synthesize chlorpyrifos photoaffinity ligand derivatives, and use these to identify proteins in THP-1 macrophages that chlorpyrifos binds to. By competition with unmodified chlorpyrifos, we will identify targets of specific binding by chlorpyrifos. We will also use CRISPR Cas9 gene editing to eliminate the protein target in THP-1 cells, and test whether this prevents chlorpyrifos binding and activation by chlorpyrifos. Results will be confirmed in experiments using human lung macrophages obtained by digestion of whole lungs. Specific Aim #2: Use genome-wide CRISPR Cas9 gene editing to eliminate proteins in THP-1 cells, testing the ability of deletions to block activation of THP-1 macrophages by chlorpyrifos. This approach will be complementary to the photocrosslinking approach in Specific Aim #1, in that it will identify both chlorpyrifos- binding proteins and also other proteins involved in signal transduction of chlorpyrifos activation. Comparing results with binding data in Specific Aim #1 will differentiate among these groups of protein targets. We will also test whether deleting the chlorpyrifos target protein decreases macrophage activation by the structurally related organophosphates diazinon and parathion, which also cause hyperreactivity by activating macrophages. These results will be confirmed in experiments using human lung macrophages obtained by digestion of whole lungs. At the completion of this project, we will have identified the molecular target responsible for macrophage activation by chlorpyrifos. Understanding this mechanism will suggest new therapeutic targets in pesticide induced asthma. It will also be relevant to the central nervous system effects of low-level organophosphate exposure, which are associated with activation of microglia, the resident macrophages of the brain. We will also have established a new method for investigating interactions of organophosphates with target cells, which will have broad applicability in studying the health effects of these ubiquitous environmental contaminants.

长期接触低浓度的有机磷杀虫剂会增加患哮喘的风险。我们 在实验动物中表明，低水平暴露会激活肺巨噬细胞产生 TNFa， 进而降低气道上抑制性 M2 毒蕈碱受体的表达和功能 副交感神经，增加乙酰胆碱的释放并增强反射性支气管收缩。 我们假设有机磷酸酯负责激活巨噬细胞的分子靶标 可以识别。为此，我们提出两个具体目标： 具体目标#1：使用光交联和点击化学来识别毒死蜱的潜在目标 与巨噬细胞结合。我们将合成毒死蜱光亲和配体衍生物，并用它们来 鉴定毒死蜱结合的 THP-1 巨噬细胞中的蛋白质。通过与未改性毒死蜱的竞争， 我们将确定毒死蜱特异性结合的目标。我们还将使用 CRISPR Cas9 基因编辑来消除 THP-1 细胞中的蛋白质靶点，并测试这是否会阻止毒死蜱的结合和激活。 结果将在使用通过消化整个肺获得的人肺巨噬细胞的实验中得到证实。 具体目标#2：使用全基因组 CRISPR Cas9 基因编辑消除 THP-1 细胞中的蛋白质，并进行测试 删除阻止毒死蜱激活 THP-1 巨噬细胞的能力。这种方法将 与具体目标 #1 中光交联方法的补充，因为它将识别毒死蜱- 结合蛋白以及参与毒死蜱激活信号转导的其他蛋白。比较 具体目标 #1 中结合数据的结果将区分这些蛋白质靶标组。我们还将 测试删除毒死蜱靶蛋白是否会通过结构相关的结构降低巨噬细胞活化 有机磷酸酯二嗪磷和对硫磷，也会通过激活巨噬细胞引起高反应性。这些 结果将在使用通过消化整个肺获得的人肺巨噬细胞的实验中得到证实。 该项目完成后，我们将确定负责巨噬细胞的分子靶标 被毒死蜱激活。了解这一机制将为农药的新治疗靶点提供建议 诱发哮喘。它还与低水平有机磷对中枢神经系统的影响有关 暴露，这与大脑常驻巨噬细胞小胶质细胞的激活有关。我们还将 建立了一种研究有机磷酸酯与靶细胞相互作用的新方法，这将 在研究这些普遍存在的环境污染物对健康的影响方面具有广泛的适用性。