Identifying the molecular target for macrophage activation by chlorpyrifos

长期接触低水平的有机磷农药会增加患哮喘的风险。我们 已经在实验动物中表明，低水平的暴露会激活肺巨噬细胞产生TNFa， 进而减少呼吸道M2受体的表达和功能 副交感神经，增加乙酰胆碱的释放，增强反射性支气管收缩。 我们假设，负责有机磷激活巨噬细胞的分子靶点 可以辨认出来。为此，我们提出了两个具体目标： 具体目标#1：使用光交联和点击化学来识别毒死蜱的潜在靶标 结合在巨噬细胞上。我们将合成毒死蜱的光亲和配体衍生物，并将其用于 识别毒死蜱结合的THP-1巨噬细胞中的蛋白质。通过与未经修饰的毒死蜱竞争， 我们将确定毒死蜱特异结合的靶标。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，并测试这是否可以阻止毒死蜱的结合和毒死蜱的激活。 结果将在使用通过消化全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基因编辑来消除THP-1细胞中的蛋白质，测试 缺失阻断毒死蜱激活THP-1巨噬细胞的能力。这种方法将是 与光交联法在特定目的#1中的补充，因为它将识别毒死蜱- 结合蛋白和其他参与毒死蜱激活的信号转导的蛋白。比较 在特定目标#1中结合数据的结果将在这些蛋白质靶标组中区分开来。我们还将 测试删除毒死蜱目标蛋白是否会通过结构上相关的 有机磷酸盐二氮磷和对硫磷，也会通过激活巨噬细胞而引起高反应性。这些 结果将在使用通过消化全肺获得的人肺巨噬细胞的实验中得到证实。 在这个项目完成时，我们将确定与巨噬细胞有关的分子靶点。 毒死蜱激活。了解这一机制将为农药提供新的治疗靶点。 诱发性哮喘。它也将与低水平有机磷对中枢神经系统的影响有关 暴露与小胶质细胞的激活有关，小胶质细胞是大脑中驻留的巨噬细胞。我们还将 已经建立了一种新的方法来研究有机磷与靶细胞的相互作用，这将 在研究这些无处不在的环境污染物对健康的影响方面具有广泛的适用性。

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.