Identifying the molecular target for macrophage activation by chlorpyrifos

长期接触低水平的有机磷农药与哮喘风险增加有关。我们 已经在实验动物中表明，低水平暴露激活肺巨噬细胞产生TNF α， 从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和增强反射性支气管收缩。 我们推测，有机磷激活巨噬细胞的分子靶点 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确定毒死蜱的潜在目标， 与巨噬细胞结合。我们将合成毒死蜱的光亲和配体衍生物，并利用这些衍生物， 鉴定THP-1巨噬细胞中与毒死蜱结合的蛋白质。通过与未改性的毒死蜱竞争， 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细胞中的蛋白质靶向，并测试这是否阻止毒死蜱结合和毒死蜱激活。 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，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 补充具体目标#1中的光交联方法，因为它将识别毒死蜱- 结合蛋白以及其他参与毒死蜱激活信号转导的蛋白。比较 具有特定目标#1中的结合数据的结果将区分这些蛋白质靶标组。我们还将 测试是否删除毒死蜱靶蛋白减少巨噬细胞激活的结构相关的 有机磷二嗪磷和二嗪磷，它们也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。 在这个项目完成后，我们将确定负责巨噬细胞的分子靶点。 被毒死蜱激活了解这一机制将为农药的开发提供新的治疗靶点 诱发哮喘这也将是有关中枢神经系统的影响，低水平的有机磷 暴露，这与小胶质细胞的激活有关，小胶质细胞是大脑的常驻巨噬细胞。我们还将 已经建立了一种新的方法来研究有机磷酸酯与靶细胞的相互作用， 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.