Mechanisms underlying metal nanoparticle-induced lung injury and fibrosis

金属纳米粒子已广泛应用于化妆品、医药、电子和工业，并且职业 或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Co、纳米Ni和纳米TiO 2）作为“模型”金属纳米颗粒来检查 它们诱导肺损伤和纤维化的能力以及所涉及的潜在潜在机制。一个 炎性小体是一种多蛋白复合物，作为caspase-1依赖性蛋白水解的平台， 成熟和分泌白细胞介素-1 β（IL-1β）。炎性小体的中心成分是 NLRP家族，该蛋白与衔接蛋白ASC相关，后者反过来招募促炎性细胞因子。 半胱天冬酶前体（例如半胱天冬酶原-1）。在许多炎性小体中，NLRP 3炎性小体是 最广泛的研究。我们的工作假设是，暴露于金属纳米粒子会导致 肺泡巨噬细胞（AM）中炎性小体和IL-1β分泌的激活和/或失调， 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 （MMPs）和金属蛋白酶的组织抑制剂（TIMPs），启动和促进金属纳米颗粒- 导致肺损伤和纤维化。该项目将使用体外和体内系统来解决 （1）确定炎性小体激活在由IL-1β诱导的IL-1β分泌中的作用。 金属纳米颗粒在体外和体内。我们将确定是否涉及炎性小体的激活 在金属纳米颗粒诱导的肺泡巨噬细胞（AM）、肺上皮细胞和肺中的IL-1β分泌中， 通过：（1）使用ac-YVAD-cmk，一种特定的半胱天冬酶-1抑制剂;（2）敲低其中一种， （3）通过使用CRISPR/Cas9技术，将炎性体组分如NLRP 3、ASC或半胱天冬酶-1结合到细胞中; 使用NLRP 3或ASC敲除小鼠。然后我们将确定NADPH氧化酶-和/或线粒体- 金属纳米颗粒诱导的炎性小体中涉及依赖性ROS产生和钾外流 活化和IL-1β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。（二） 研究IL-1β对肺细胞MMPs和TIMPs表达和活性的影响 暴露在金属纳米颗粒中虽然IL-1β是MMP-2和MMP-9活性的诱导剂，但其作用机制尚不清楚。 通过暴露于金属纳米颗粒调节MMP和TIMP。为了检测炎性小体和IL-12的作用， 1β在MMPs和TIMPs调节中的作用，目的1中的策略将用于抑制炎性小体功能， 抑制IL-1功能的策略将通过使用：（1）药理学IL-1β抑制剂;（2）抗IL-1 β抗体。 1β抗体;和（3）IL-1 RI-/-小鼠，其将不响应IL-1β。在暴露于金属纳米颗粒后，MMP-2， 将测定MMP-9和TIMP的表达和活性。(3)研究炎性小体的作用 在体内金属纳米颗粒诱导的肺损伤和纤维化中的活化。我们将首先调查 暴露于金属纳米颗粒将导致肺成纤维细胞产生更多的胶原蛋白。那么， 金属纳米颗粒诱导的肺纤维化中的炎性小体将通过短期和长期暴露于 小鼠对金属纳米颗粒的反应。我们将使用目标1和目标2中的策略来抑制炎性小体和IL-1β功能 探讨炎性小体和IL-1β在金属纳米颗粒诱导的肺纤维化中的作用。

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.