PCB-induced priming of microglial activity across sexes and development

项目摘要/摘要 多氯联苯(PCbs)仍然是无处不在的环境污染物，并与之接触 与神经发育和情绪障碍的风险增加有关。这些紊乱倾向于 更多发生在婴儿男性和青春期女性个体中，并与改变有关 小胶质细胞的功能，这是大脑的常驻免疫细胞。虽然在以下方面取得了重大进展 了解多氯联苯对神经元的影响、多氯联苯的直接影响和大多数其他环境 毒物对小胶质细胞的作用尚不清楚。这项建议的总体目标是确定直接影响 以及多氯联苯对小胶质细胞功能和可能的神经元后果的可能作用机制。 中心假设是，多氯联苯将启动小胶质细胞，以产生夸大的反应 在年龄和性别方面的次要挑战。该应用程序建议敏锐地使用小胶质细胞 从男女新生儿和青少年的全脑中分离并单独治疗多氯联苯 同系物，一种具有环境代表性的混合物，或在控制或免疫之前的体外载体 挑战。目的1评价多氯联苯暴露对小胶质细胞因子信号转导的影响。 监管。采用定量聚合酶链式反应和酶联免疫吸附试验检测IL-1b和TNFa的表达和释放。表达式 细胞因子所需的促炎性TLR4受体和NFkB转录因子的定位 表达也将通过免疫细胞化学来确定。目标2是审问效果和 多氯联苯对小胶质细胞氧化应激的可能机制。ROS/RNS和过氧亚硝酸盐的生产 混合细胞培养中的相关细胞损伤以及调节的多氯联苯敏感受体的表达 小胶质细胞的钙和ROS/RNS的产生将被测定。目标3是确定影响和 多氯联苯促进小胶质细胞吞噬的可能机制。荧光小胶质细胞吞噬功能 标记的乳胶珠和吞噬相关基因的表达将通过流式细胞仪进行检测 和qPCR。将在混合细胞培养中确定与吞噬作用有关的小胶质细胞的形态 采用免疫组织化学方法。这项拟议的工作具有创新性，因为它是第一个直接研究性的工作 小胶质细胞对环境毒物反应的差异和使用急性分离技术 更好地保持体内的表型。预计这些研究将有助于形成一个概念框架。 用于识别小胶质细胞中多氯联苯作用的机制靶点。这一知识可以为我们提供强大的 旨在治疗或预防的小胶质细胞导向治疗策略的追求基础 毒物引起的神经发育障碍。这一信息还可以为以人口为目标的 减少暴露的战略。作为R15领域的应用，这些研究将使学生参与到 自主建功立业，强化院系研究环境 为大量学生提供服务的机构，这些学生在科学方面的代表性不足。

PROJECT SUMMARY / ABSTRACT Polychlorinated biphenyls (PCBs) remain ubiquitous environmental contaminants and exposure to them is associated with increased risk of neurodevelopmental and mood disorders. These disorders tend to occur more frequently in infant male and adolescent female individuals, and are associated with altered function of microglia, the resident immune cells of the brain. While significant gains have been made in understanding effects of PCBs on neurons, direct effects of PCBs and most other environmental toxicants on microglia are unknown. The overall objective of this proposal is to identify direct effects and putative mechanisms of PCB action on microglial function and possible neuronal consequences. The central hypothesis is that PCBs will prime microglia to produce exaggerated responses to a secondary challenge in age- and sex-specific ways. The application proposes to use microglia acutely isolated from male and female neonatal and adolescent whole brains and treated to individual PCB congeners, an environmentally representative mixture, or vehicle in vitro prior to a control or immune challenge. Aim 1 is to evaluate the effects of PCB exposure on microglial cytokine signaling and regulation. IL1b and TNFa expression and release will be assayed with qPCR and ELISA. Expression and localization of pro-inflammatory TLR4 receptor and NFkB transcription factor required for cytokine expression will also be determined with immunocytochemistry. Aim 2 is to interrogate effects and putative mechanisms of PCBs on microglial oxidative stress. Production of ROS/RNS and peroxynitrite- associated cell damage in mixed cell cultures, and expression of PCB-sensitive receptors that modulate calcium and ROS/RNS production in microglia, will be determined. Aim 3 is to identify effects and putative mechanisms of PCBs on microglial phagocytosis. Microglial phagocytosis of fluorescently labeled latex beads and expression of phagocytosis-related genes will be determined by flow cytometry and qPCR. Microglia morphology associated with phagocytosis will be determine in mixed cell cultures by immunohistochemistry. The proposed work is innovative in that it is the first to directly study sex differences in microglial responses to environmental toxicants and use acute isolation techniques to better maintain in vivo phenotypes. These studies are expected to contribute to a conceptual framework for identifying mechanistic targets of PCB action in microglia. This knowledge could provide a strong foundation for the pursuit of microglia-directed therapeutic strategies aimed at treating or preventing toxicant-induced neurodevelopmental disorders. This information could also inform population-targeted exposure mitigation strategies. As an AREA-R15 application, these studies will engage students in independent and meritorious research, strengthening the institutional research environment at an institution serving a large number of students underrepresented in science.