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.

长期暴露于低水平的有机磷酸盐农药与哮喘风险增加有关。我们 在实验动物中显示的低水平暴露会激活肺巨噬细胞以产生TNFA， 反过 副交感神经，增加乙酰胆碱的释放和增强反射支气管收缩。 我们假设有机磷酸盐负责激活巨噬细胞的分子靶标 可以识别。为此，我们提出了两个具体目标： 特定目的＃1：使用Photocroslinking并单击化学以识别Chlorpyrifos的潜在目标 与巨噬细胞结合。我们将综合毒性cropyapperition配体衍生物，并将其用作 鉴定毒死rif虫结合的Thp-1巨噬细胞中的蛋白质。通过与未修改的毒蛇竞争的竞争， 我们将确定毒死rif虫特异性结合的靶标。我们还将使用CRISPR CAS9基因编辑来消除 THP-1细胞中的蛋白质靶标，并测试这是否阻止了毒死rif虫的结合和毒死rif虫的激活。 在实验中，使用消化整个肺部获得的人类肺巨噬细胞将证实结果。 特定目的＃2：使用全基因组CRISPR CAS9基因编辑来消除THP-1细胞中的蛋白质，测试 缺失阻止毒死rif虫对THP-1巨噬细胞激活的能力。这种方法将是 在特定目标1中与光链接方法的互补，因为它将确定两种毒死rif- 结合蛋白以及其他参与毒死rif虫激活信号转导的蛋白质。比较 在特定目标＃1中具有结合数据的结果将在这些蛋白质靶标组中区分。我们也会 测试删除毒死rif靶蛋白是否会通过结构相关的巨噬细胞降低巨噬细胞的激活 有机磷酸盐二氮和parathion，这也通过激活巨噬细胞引起过度反应性。这些 在实验中，使用消化整个肺部获得的人类肺巨噬细胞将证实结果。 该项目完成时，我们将确定负责巨噬细胞的分子靶标 毒死rif虫的激活。了解这种机制将暗示农药的新治疗靶标 诱发哮喘。它也将与低级有机磷酸盐的中枢神经系统效应有关 暴露与小胶质细胞激活有关的暴露是大脑的驻留巨噬细胞。我们也会 已经建立了一种研究有机磷酸盐与靶细胞相互作用的新方法，这将 在研究这些无处不在的环境污染物的健康影响方面具有广泛的适用性。