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.

长期接触低水平的有机磷农药会增加患哮喘的风险。我们