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 ...