Environmental arsenic, immunoregulation, and viral disease risk

项目总结/摘要 井水和食物中的砷暴露是一个主要的公共卫生问题， 病毒感染的发病率和死亡率甲型流感病毒（IAV）每年感染约500万人， 急性呼吸道症状和多达646，000人死亡。病毒性疾病的严重程度因人而异 并且可能涉及对免疫调节的遗传和环境影响。流行病学和动物 模型研究表明，生命早期暴露于砷会改变对病原体的免疫反应， 长期或过度的炎症。此外，现有的证据表明，砷是一种内分泌 干扰糖皮质激素受体（GR）信号通路的干扰物， 炎症，可能具有代际表观遗传效应。然而，在我们的国家， 了解砷如何破坏GR信号传导并促进炎症。探索性/发展性 一个研究项目将使用斑马鱼作为模型系统来测试砷加剧病毒感染的新假设。 通过下调klf 9依赖性抗炎GR信号通路来治疗疾病。初步研究 表明Klf 9是促炎基因GR响应性负调节因子，而基础和皮质醇- 在暴露于非常低水平的砷的斑马鱼胚胎中，诱导的KLF 9活性受到抑制。斑马鱼幼体 具有功能性先天免疫系统，是研究宿主-病原体相互作用的有力模型， 全身性或局部性流感（IAV）感染，如巨噬细胞和中性粒细胞向IAV部位的运输 感染也可以使用具有荧光标记的白细胞的转基因系的活体成像来可视化 荧光标记的病毒。拟议的研究将使用这些工具，以及GR和klf 9敲除 我们最近使用CRISPR创建的线，以实现两个特定的目标。首先是确定砷是否 通过抑制抗炎性GR-Klf 9信号传导来失调对IAV感染的炎症反应 途径，导致过度和/或延长的促炎基因表达和未能解决 反应这将通过询问用砷处理斑马鱼幼虫如何影响表达来实现 klf 9和下游促炎基因，我们已经确定为Klf 9介导的炎症反应的假定靶点。 抑制，并评估砷和klf 9剂量对炎症细胞反应动力学的影响 （中性粒细胞和巨噬细胞）和NF-kB活性。第二个具体目标是 确定砷暴露是否对IAV感染的先天免疫反应具有代际影响， 与GR-Klf 9免疫调节途径的异常活性相关。为了实现这一点，砷-或 将暴露于溶剂的野生型幼虫饲养至成年，并通过2代近交，而不进一步 exposure.在每一代（F1和F2）的幼虫中，我们将评估免疫调节基因的表达， IAV感染后的幼虫存活率。该项目将阐明一种新的抗病毒免疫调节途径， 通过砷，开辟了一条途径，为未来的研究重点是进一步阐明潜在的机制。

PROJECT SUMMARY / ABSTRACT Arsenic exposure from well water and food is a major public health concern and is associated with increased morbidity and mortality from viral infections. Influenza A virus (IAV) annually infects ~5 million people causing acute respiratory symptoms and up to 646,000 deaths. The severity of viral disease varies between individuals and is likely to involve both genetic and environmental effects on immunoregulation. Epidemiological and animal model studies suggest that early life exposure to arsenic alters the immune response to pathogens, resulting in prolonged or excessive inflammation. Furthermore, available evidence indicates that arsenic is an endocrine disruptor that interferes with the glucocorticoid receptor (GR) signaling pathway, a critical regulator of inflammation, possibly with intergenerational epigenetic effects. However, significant gaps remain in our understanding of how arsenic disrupts GR signaling and promotes inflammation. This exploratory/ developmental research project will use zebrafish as a model system to test the novel hypothesis that arsenic exacerbates viral disease by downregulating the klf9-dependent anti-inflammatory GR signaling pathway. Preliminary studies indicate that Klf9 is a GR-responsive negative regulator of proinflammatory genes, and that basal and cortisol- induced klf9 activity is suppressed in zebrafish embryos exposed to very low levels of arsenic. Zebrafish larvae have a functional innate immune system and are a powerful model to study host-pathogen interactions during systemic or localized influenza (IAV) infection, as trafficking of macrophages and neutrophils to the site of IAV infection can be visualized using live imaging with transgenic lines with fluorescently labeled leukocytes as well as fluorescently labeled viruses. The proposed research will use those tools, as well as GR amd klf9 knockout lines that we recently created using CRISPR, to accomplish two specific aims. The first is to determine if arsenic dysregulates the inflammatory response to IAV infection by suppressing the anti-inflammatory GR-Klf9 signaling pathway, leading to excessive an/or prolonged pro-inflammatory gene expression and failure to resolve the response. This will be accomplished by asking how treatment of zebrafish larvae with arsenic affects expression of klf9 and downstream proinflammatory genes that we have identified as putative targets of Klf9-mediated repression, and assessing the effects of arsenic and klf9 dosage on the response dynamics of inflammatory cells (neutrophils and macrophages) and NF-kB activity following IAV infection. The second specific aim is to determine if arsenic exposure has intergenerational effects on the innate immune response to IAV infection that correlate with aberrant activity of the GR-Klf9 immunoregulatory pathway. To accomplish this, F0 arsenic- or vehicle-exposed wild-type larvae will be raised to adulthood and inbred through 2 generations without further exposure. In larvae from each generation (F1 and F2) we will assess immunoregulatory gene expression and larval survival after IAV infection. The project will elucidate a novel anti-viral immunoregulatory pathway impacted by arsenic, opening an avenue for future research focused on further elucidating the underlying mechanisms.