PCB-induced priming of microglial activity across sexes and development

项目总结/摘要 多氯联苯（PCBs）是普遍存在的环境污染物， 与神经发育和情绪障碍的风险增加有关。这些疾病往往 更频繁地发生在婴儿男性和青春期女性个体中，并且与改变的 小胶质细胞的功能，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影响 对小胶质细胞的毒物是未知的。本提案的总体目标是确定直接影响 以及PCB对小胶质细胞功能和可能的神经元后果的作用的推定机制。 核心假设是，多氯联苯将引发小胶质细胞对一种刺激产生夸张的反应。 在特定年龄和性别方面的次要挑战。该申请建议使用小胶质细胞急性 从男性和女性新生儿和青少年的全脑中分离，并对个体PCB进行处理 在对照或免疫前， 挑战.目的1是评估PCB暴露对小胶质细胞细胞因子信号传导的影响， 调控将使用qPCR和ELISA测定IL 1b和TNF α的表达和释放。表达 以及细胞因子所需的促炎性TLR 4受体和NFkB转录因子的定位 表达也将用免疫细胞化学测定。目标2是询问效果， PCBs对小胶质细胞氧化应激的可能机制ROS/RNS和过氧亚硝酸盐的产生- 在混合细胞培养物中相关的细胞损伤，以及调节PCB敏感受体的表达， 钙和ROS/RNS的产生。目标3是确定影响， PCBs对小胶质细胞吞噬作用的推测机制。小胶质细胞吞噬荧光 标记的乳胶珠和吞噬相关基因的表达将通过流式细胞术测定 qPCR。将在混合细胞培养物中确定与吞噬作用相关的小胶质细胞形态 应用免疫这项工作的创新之处在于它是第一个直接研究性的 小胶质细胞对环境毒物反应的差异，并使用急性分离技术， 更好地维持体内表型。预计这些研究将有助于建立一个概念框架 用于识别PCB在小胶质细胞中作用的机制目标。这些知识可以提供一个强大的 小胶质细胞导向治疗策略基金会 毒物引起的神经发育障碍这些信息还可以为针对人口的 暴露缓解策略。作为AREA-R15应用程序，这些研究将让学生参与 独立和有价值的研究，加强机构研究环境， 一所为大量理科学生提供服务的机构。

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.