Environmental arsenic, immunoregulation, and viral disease risk

项目摘要/摘要 从井水和食物中接触到砷是一个主要的公共卫生问题，并与 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，导致 急性呼吸道症状和高达64.6万人死亡。病毒性疾病的严重程度因人而异。 而且很可能涉及免疫调节的遗传和环境影响。流行病学和动物学 模型研究表明，早年接触砷会改变对病原体的免疫反应，导致 长期的或过度的炎症。此外，现有证据表明，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，糖皮质激素受体(GR)信号通路的关键调节因子 炎症，可能具有代际表观遗传效应。然而，在我们的 了解砷是如何扰乱GR信号并促进炎症的。这种探索性/发展性 研究项目将使用斑马鱼作为模型系统来测试砷会加剧病毒这一新假设 通过下调KLF9依赖的抗炎GR信号通路而致病。初步研究 提示KLF9是促炎症基因的GR反应性负调控因子，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KLF9活性受到抑制。斑马鱼幼体 有一个正常运作的先天免疫系统，是研究宿主-病原体相互作用的强大模型 系统性或局部性流感(IAV)感染，如巨噬细胞和中性粒细胞向IAV部位运输 也可以使用带有荧光标记的白细胞的转基因系的活体成像来显示感染情况 作为荧光标记的病毒。拟议的研究将使用这些工具，以及GR和KLF9基因敲除 我们最近使用CRISPR创建的线路，以实现两个特定目标。第一个是确定砷是否 通过抑制抗炎因子GR-Klf9信号转导途径调节机体对IAV感染的炎症反应 途径，导致过多的AN/或延长促炎基因的表达，并无法解决 回应。这将通过询问用砷处理斑马鱼幼体如何影响表达来实现。 KLF9及其下游的致炎基因，我们已经确定它们是KLF9介导的假定靶点 抑制，并评价砷和KLF9剂量对炎性细胞反应动力学的影响 (中性粒细胞和巨噬细胞)和核因子-kB的活性。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有代际影响 与GR-KLF9免疫调节通路的异常活性有关。要做到这一点，F0砷-或 车辆暴露的野生型幼虫将被培养到成体，并通过2代近亲交配，不需要进一步的 曝光。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和 幼虫在IAV感染后存活。该项目将阐明受影响的一种新的抗病毒免疫调节途径 通过砷，为未来的研究开辟了一条途径，重点是进一步阐明其潜在机制。

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.