Mechanisms underlying metal nanoparticle-induced lung injury and fibrosis

金属纳米颗粒诱导肺损伤和纤维化的机制

• 批准号: 9794949
• 负责人: Qunwei Zhang
• 金额: $ 38.96万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9794949
• 关键词: Adaptor Signaling Protein; Address; Alveolar Macrophages; Antibodies; Apoptosis; Asbestos; Asthma; Attenuated; Biological; Bronchoalveolar Lavage Fluid; CASP1 gene; CRISPR/Cas technology; Caspase; Cells; Collagen; Cosmetics; Development; Electronics; Epithelial Cells; Exposure to; Family; Fibroblasts; Fibrosis; Gelatinase A; Gelatinase B; Generations; Health; In Vitro; Industrialization; Industry; Inflammasome; Inflammatory; Injury; Interleukin-1; Interleukin-1 Receptors; Interleukin-1 beta; Knockout Mice; Lead; Lesion; Lung; Lung Inflammation; Lung diseases; Malignant neoplasm of lung; Matrix Metalloproteinases; Measures; Medical; Medicine; Metal exposure; Metal fever; Metals; Mitochondria; Modeling; Multiprotein Complexes; Mus; NADPH Oxidase; Normal Range; Occupational; Pharmacology; Play; Potassium; Proteins; Pulmonary Fibrosis; Regulation; Role; Silicon Dioxide; System; Testing; Tissue Inhibitor of Metalloproteinases; Toxic effect; Transition Elements; Wild Type Mouse; anakinra; base; exposed human population; in vivo; inhibitor/antagonist; interest; interleukin-1beta-converting enzyme inhibitor; knock-down; lung injury; macrophage; member; monocyte; nano; nanomaterials; nanoparticle; particle; recruit; response; titanium dioxide; tool

Metal nanoparticles have been widely used in cosmetics, medicine, electronics, and industry, and occupational or non-occupational exposure to metal nanoparticles is growing. In this proposal, we have selected several transition metal nanoparticles (Nano-Co, Nano-Ni, and Nano-TiO2) as `model' metal nanoparticles to examine their ability to induce pulmonary injury and fibrosis and the potential underlying mechanisms involved. An inflammasome is a multiprotein complex that serves as a platform for caspase-1-dependent proteolytic maturation and secretion of interleukin-1β (IL-1β). The central component of an inflammasome is a member of the NLRP family, and this protein associates with the adaptor protein ASC, which in turn recruits pro-inflammatory caspase precursors (such as procaspase-1). Among a number of inflammasomes, the NLRP3 inflammasome is the most extensively studied. Our working hypothesis is that exposure to metal nanoparticles will cause activation and/or dysregulation of the inflammasome and IL-1β secretion in alveolar macrophages (AMs), lung epithelial cells, and lung fibroblasts, which will cause dysregulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), initiating and promoting metal nanoparticle- induced pulmonary injury and fibrosis. This project will use both in vitro and in vivo systems to address the following specific aims: (1) Determine the role of inflammasome activation in IL-1β secretion induced by metal nanoparticles in vitro and in vivo. We will identify whether activation of the inflammasome is involved in metal nanoparticle-induced IL-1β secretion in alveolar macrophages (AMs), lung epithelial cells, and lung fibroblasts by: (1) using ac-YVAD-cmk, a particular inhibitor of caspase-1; (2) knocking-down one of the inflammasome components such as NLRP3, ASC, or caspase-1 by using CRISPR/Cas9 technology; and (3) using NLRP3 or ASC knock-out mice. We will then determine whether NADPH oxidase- and/or mitochondria- dependent ROS generation and potassium efflux are involved in metal nanoparticle-induced inflammasome activation and IL-1β secretion. We will also measure IL-1β secretion in mice exposed to metal nanoparticles. (2) Examine the role of IL-1β in the alteration of MMPs and TIMPs expression and activity in lung cells exposed to metal nanoparticles. While IL-1β is an inducer for MMP-2 and MMP-9 activity, it is unclear how it regulates MMPs and TIMPs with exposure to metal nanoparticles. To test the role of the inflammasome and IL- 1β in the regulation of MMPs and TIMPs, the strategies in Aim 1 will be used to inhibit inflammasome function, and strategies to inhibit IL-1 function will be applied by using: (1) the pharmacologic IL-1β inhibitor; (2) anti-IL- 1β antibody; and (3) IL-1RI-/- mice that will not respond to IL-1β. After exposure to metal nanoparticles, MMP-2, MMP-9 and TIMPs expression and activity will be determined. (3) Investigate the role of inflammasome activation in metal nanoparticle-induced lung injury and fibrosis in vivo. We will first investigate whether exposure to metal nanoparticles will cause lung fibroblasts to produce more collagen. Then the role of the inflammasome in metal nanoparticle-induced lung fibrosis will be explored by short- and long-term exposure of mice to metal nanoparticles. We will use the strategies in Aim 1 and 2 to inhibit inflammasome and IL-1β function to investigate the role of the inflammasome and IL-1β in metal nanoparticle-induced lung fibrosis.

金属纳米颗粒已广泛应用于化妆品、医药、电子、工业和职业领域。 或者，非职业性接触金属纳米颗粒的情况正在增加。在这份提案中，我们选择了几个 过渡金属纳米颗粒(纳米钴、纳米镍和纳米二氧化钛)作为要检验的“模型”金属纳米颗粒 它们诱导肺损伤和纤维化的能力以及涉及的潜在潜在机制。一个 炎症体是一种多蛋白复合体，可作为caspase-1依赖的蛋白分解的平台。 白介素1β(IL-1β)的成熟和分泌。炎性小体的中心组件是 NLRP家族，这种蛋白与接头蛋白ASC结合，ASC反过来招募促炎因子 Caspase前体(如proaspase-1)。在许多炎症体中，NLRP3炎症体是 研究最广泛的。我们的工作假设是暴露在金属纳米颗粒中会导致 肺泡巨噬细胞(AM)炎症小体和IL-1β分泌的激活和/或调节失调， 肺上皮细胞和肺成纤维细胞，这将导致基质金属蛋白酶的失调 金属蛋白酶(MMPs)和金属蛋白酶组织抑制物(TIMPs)，引发和促进金属纳米颗粒- 致肺损伤和纤维化。该项目将使用体外和体内系统来解决 具体目的如下：(1)确定炎性小体激活在诱导IL-1β分泌中的作用 金属纳米粒子的体外和体内研究。我们将确定是否涉及炎性小体的激活 金属纳米颗粒诱导肺泡巨噬细胞、肺上皮细胞和肺组织中IL-1β的分泌 通过：(1)使用caspase-1的特定抑制剂Ac-YVAD-CMK；(2)击倒其中一个 使用CRISPR/Cas9技术的炎症体成分，如NLRP3、ASC或caspase-1；以及(3) 使用NLRP3或ASC基因敲除小鼠。然后我们将确定NADPH氧化酶-和/或线粒体- 依赖的ROS生成和钾外流参与了金属纳米颗粒诱导的炎性小体 活化和IL-1β的分泌。我们还将测量暴露于金属纳米颗粒的小鼠的IL-1β分泌。(2) 检测IL-1β在肺细胞基质金属蛋白酶及其活性变化中的作用 暴露在金属纳米颗粒中。尽管IL-1β是基质金属蛋白酶-2和基质金属蛋白酶-9活性的诱导剂，但目前还不清楚它是如何 通过暴露于金属纳米颗粒来调节MMPs和TIMP。为了测试炎症小体和IL-2的作用 1β在MMPs和TIMPs的调节中，目标1中的策略将用于抑制炎症小体功能， 而抑制IL-1功能的策略将通过使用：(1)药理IL-1β抑制剂；(2)抗IL-1- 1β抗体；(3)对IL-1β无反应的IL-1RI-/-小鼠。在接触到金属纳米颗粒，基质金属蛋白酶-2， 将测定MMP9和TIMPs的表达和活性。(3)探讨炎性小体的作用 在体内金属纳米颗粒诱导的肺损伤和纤维化中的激活。我们将首先调查是否 暴露于金属纳米颗粒会导致肺成纤维细胞产生更多的胶原。那么这个角色是 金属纳米颗粒诱导的肺纤维化中的炎症体将通过短期和长期暴露于 从老鼠到金属纳米粒子。我们将使用目标1和2中的策略来抑制炎症体和IL-1β功能 探讨炎性小体和IL-1β在金属纳米颗粒诱导肺纤维化中的作用。