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得克隆602和二恶英 的肠道免疫效应及机理探讨
Alternative TitleEffects of Dechlorane 602 and dioxin on gut immunity and preliminary study of involved mechanisms
李云平
Subtype博士
Thesis Advisor赵斌
2019-12
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
KeywordTcdd,得克隆602,结肠炎症,iii型固有淋巴细胞,微生物 tcdd, Dec 602, Colonic Inflammation, Ilc3s, Microbiota
Abstract

      二恶英等典型的持久性有机污染物 persistent organic pollutants, POPs 具有复杂的免疫毒性,可造成获得性免疫系统和固有免疫系统的多种毒性效应。肠道不仅是消化和吸收营养物质的场所,同时也是一个重要的免疫器官。 肠道中有大量共生微生物,当 肠道微生物的稳态遭到破坏时,也会对免疫反应产生影响,甚至导致肠道炎症的发生。肠道免疫功能及其与肠道微生物的稳态关系共同构成机体响应 POPs食源性暴露的 重要 防御屏障 。 因此明确 POPs对肠道免疫稳态的影响具有重要的毒理学意义。
      食物是二恶英人体暴露的主 要途径。 2,3,7,8-四氯二苯并对二恶英( 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD 是 毒性最强的二恶英类化合物 具有免疫毒性和遗传毒性,但是 TCDD对肠道免疫系统,特别是对肠道固有免疫的垂直干扰效应尚未明确。得克隆是一类氯代阻燃剂,其中得克隆 602 Dechlorane 602, Dec 602 在 环境 中的 检出浓度相对较高,并具有明确的生物累积和放大效应,是潜在的新型 POPs。 人体 每天 通过 肉类、鱼类和蛋类等食物 摄入的 Dec 602可达 463 pg 说明食源性暴露也是 Dec 602进入 人体的重要途径, 然而关于 Dec 602对肠道免疫系统和肠道共生微生物影响的研究尚未见报道。
      针对以上问题,本研究选取TCDD和 Dec 602作为 研究对象, 以 C57BL/6小鼠为动物模型, 研究污染物 在体暴露 对肠道免疫,特别是对一种新型固有免疫细胞,即 III型固有淋巴细胞( type III innate lymphoid cells, ILC3s 的功能 与分化的 干扰效应,以及 Dec 602对肠道免疫和共生微生物的影响 并探讨 了 污染物的作用机制 。 取得了 一下 研究 结果
      (1 )发现 Dec 602亚急性暴露 后, 小鼠结肠发生炎症,肠道 免疫系统与 共生 微生物之间的 稳态被打破,同时破坏了 Treg/Th17细胞的免疫平衡 。 具体 表现为 Dec 602 1 μg/kg BW和 10 μg/kg BW 连续暴露 7天后 小鼠结肠组织固有层出现炎症性淋巴细胞浸润 ;细胞与免疫分子分析显示 ,结肠固有层 CD4+ T细胞对 白介素 -22 interleukin-22, IL-22 的分泌水平降低, IL-22+ ILC3s固有淋巴细胞比例降低 肠系膜淋巴结中 起 炎症抑制作用的 Treg细胞比例减少 同时发现与炎症相关的菌群丰度升高 。
      (2) 终止 Dec 602暴露 7天 后 小鼠结肠炎症没有恢复,肠道免疫系统与共生微生物仍然处于 炎症 状态,而视黄酸信号通路在这一过程中发挥着 一定 作用 。具体表现为 Dec 602 1 μg/kg BW和 10 μg/kg BW 连续暴露终止 7天后小鼠结肠固有层 仍然有明显的 淋巴细胞浸润 现象 ,结肠固有层 ILC3s细胞 的 IL-22细胞因子 表达 降低 肠系膜淋巴结中 CD4+ T细胞比例增加 同时 有益菌群丰度降低,与炎症相关的菌群丰度增加 。结肠转录组分析发现, 在 Dec 602暴露 终止 7天后 上皮 -间充质转换和血管生成相关基因上调 并发现 与肠道免疫稳态 有关的 视黄酸核受体 RARα的转录 水平降低 提示 视黄酸信号通路 的 扰动可能参与
Dec 602诱导 的肠道炎症 。
      (3) 发现 TCDD母 体暴露可以对 子 鼠结肠固有层 ILC3s细胞的组成和功能因子分泌产生干扰作用, 证明 TCDD对 ILC3s的 发育与功能存在垂直干扰作用,而这种作用在分离细胞体外直接暴露 的 条件下并不存在 。 具体表现为 TCDD 30 μg/kg BW 7天 短期暴露后 成年 小鼠结肠固有层中 NKp46+ ILC3s细胞和淋巴组织诱导细胞比例均显著降低 对孕鼠进行 TCDD 0.1 μg/kg BW和 10 μg/kg BW 10-15周)暴露后,子鼠结肠中 ILC3s细胞比例显著降低, NKp46+ ILC3s细胞比例显著升高 IL-17a+ ILC3s细胞比例在子鼠和母鼠体内均显著升高 IFNγ+ ILC3s细胞比例只在母鼠体内显著升高; 而 TCDD 0.1-10 nmol/L 体外 处理分离的结肠固有层淋巴细胞 2天 后,并没有发现 ILC3s细胞亚群的比例的变化。
本研究表明, TCDD和 Dec 602对肠道免疫稳态可以造成短期和 持续性的干扰为食源性有机污染物的肠道免疫毒性研究提供了新的数据和 方法借鉴 。

Other Abstract

      Persistent organic pollutants (POPs), such as dioxins, have various effects on adaptive and innate immunities. Gut not only has the function of nutrient absorption, but also is an important organ for immunity. There are trillions of microbes living in the gut, and the imbalance of gut microbiota will influence the immune responses, including adaptive and innate immunities and lead to intestinal inflammation. The homeostasis of gut immunity and microbiota composes the first line of defense against foodborne POPs exposure. Thus, it is important to reveal the effects of these POPs on gut immunity.
      Contaminated food intake is one of the principal ways to expose to POPs in human. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic dioxins which has genetic and immune toxicity. However, little is known about the vertical interference of TCDD on the function and development of gut innate immunity. Dechloranes are chlorinated flame retardants. Among them, environmental concentrations of dechlorane 602 (Dec 602) are higher than the others. Dec 602 has biomagnification and bioaccumulation capacity, which suggests that it may be a potential POPs. Human can intake 463 pg/day Dec 602 through contaminated food, such as meat, fish and eggs, which suggests foodborne exposure is an important way for Dec 602. Nonetheless, there are few reports about the effects of Dec 602 on gut immunity and microbiota.
      Therefore, in this study, C57BL/6 mice were exposed to TCDD and Dec 602 in vivo to study the interferences of these compounds on the gut immunity, especially on type III innate lymphoid cells (ILC3s), and the effects of Dec 602 on gut immunity and microbiota. Underlining mechanisms for Dec 602 effects on gut immunity were also explored preliminarily. We got the following results:

      (1) Subacute exposure to Dec 602 induced colonic inflammation, disturbed the homeostasis of gut immunity and microbiota, and influenced the balance of Treg and Th17 immune responses. After seven consecutive days of Dec 602 (1 μg/kg BW and 10 μg/kg BW) exposure, histological studies showed that there was infiltration of inflammatory lymphocytes in the colonic lamina propria; studies on the immune cells and molecules showed that production of interleukin (IL)-22 by CD4+ T cells and percentage of IL-22+ ILC3s in lamina propria were decreased, as well as Treg cells in mesenteric lymph nodes which can suppress inflammation; meanwhile, the gut microbiota involved in inflammation was increased.
       (2) In the long-lasting effect study, even 7 days after ceasing exposure to Dec 602, we still found the colonic inflammation remain histologically, and the inflammatory state of gut immunity and microbiota remain as well, in which alteration in retinoic acid pathway activity may be involved. The infiltration of lymphocytes was found in the colonic lamina propria and the suppressive expression of IL-22 was observed in colonic ILC3s. However, the percentage of CD4+ T cells was increased in mesenteric lymph nodes of mice with Dec 602 exposure. In gut, probiotics was decreased, and microbiota involved in inflammation was still increased. In addition, 7 days after ceasing Dec 602 exposure, genes related to the epithelial-mesenchymal transition and blood vessel formation were upregulated. The mRNA level of nuclear receptor of retinoic acid, RARα, was downregulated which suggested that the disturbation of retinoic acid pathway may be involved in the induction of colonic inflammation.
       (3) Maternal exposure of TCDD influenced the composition of ILC3s and production of ILC3s effector cytokines. Meanwhile, vertical interference of TCDD was found on the development and fuctions of ILC3s in the offspring, which was not observed after treatment with TCDD in cultured isolated lamina propria lymphocytes in vitro. After short-term exposure to TCDD (30 μg/kg BW, 7 days), the percentages of NKp46+ ILC3s and lymphoid tissue-inducer (LTi) cells were decreased in the colonic lamina propria of the adult mice; while the long-term maternal exposure to TCDD (0.1 μg/kg BW and 10 μg/kg BW, 10-15 weeks) induced the upregulation of NKp46+ ILC3s and downregulation of total ILC3s in the colonic lamina propria in the offspring. The percentage of IL-17a+ ILC3s was increased in the offspring and mother, but the percentage of IFN-γ+ ILC3s was increased only in the mother. Treatment with TCDD (0.1-10 nmol/L) in lamina propria lymphocytes in vitro did not influence the percentages of these ILC3s subsets.
       In conclusion, exposure to TCDD or Dec 602 can induce short-term and long-lasting interferences on the homeostasis of gut immunity, which provides new data and approaches to study the gut immune toxicity of organic pollutants via foodborn exposure.

Pages165
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42242
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
李云平. 得克隆602和二恶英 的肠道免疫效应及机理探讨[D]. 北京. 中国科学院生态环境研究中心,2019.
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