The Effect of Size on Ag Nanosphere Toxicity in Macrophage Cell Models and Lung Epithelial Cell Lines Is Dependent on Particle Dissolution

Silver (Ag) nanomaterials are increasingly used in a variety of commercial applications. This study examined the effect of size (20 and 110 nm) and surface stabilization (citrate and PVP coatings) on toxicity, particle uptake and NLRP3 inflammasome activation in a variety of macrophage and epithelial cell lines. The results indicated that smaller Ag (20 nm), regardless of coating, were more toxic in both cell types and most active in the THP-1 macrophages. TEM imaging demonstrated that 20 nm Ag nanospheres dissolved more rapidly than 110 nm Ag nanospheres in acidic phagolysosomes consistent with Ag ion mediated toxicity. In addition, there were some significant differences in epithelial cell line in vitro exposure models. The order of the epithelial cell lines’ sensitivity to Ag was LA4 \u3e MLE12 \u3e C10. The macrophage sensitivity to Ag toxicity was C57BL/6 AM \u3e MARCO null AM, which indicated that the MARCO receptor was involved in uptake of the negatively charged Ag particles. These results support the idea that Ag nanosphere toxicity and NLRP3 inflammasome activation are determined by the rate of surface dissolution, which is based on relative surface area. This study highlights the importance of utilizing multiple models for in vitro studies to evaluate nanomaterials

The Effects of Size on Ag Nanosphere Toxicity in Macrophage Cell Models and Lung Epithelial Cell Lines Is Dependent on Particle Dissolution

Silver (Ag) nanomaterials are increasingly used in a variety of commercial applications. This study examined the effect of size (20 and 110 nm) and surface stabilization (citrate and PVP coatings) on toxicity, particle uptake and NLRP3 inflammasome activation in a variety of macrophage and epithelial cell lines. The results indicated that smaller Ag (20 nm), regardless of coating, were more toxic in both cell types and most active in the THP-1 macrophages. TEM imaging demonstrated that 20 nm Ag nanospheres dissolved more rapidly than 110 nm Ag nanospheres in acidic phagolysosomes consistent with Ag ion mediated toxicity. In addition, there were some significant differences in epithelial cell line in vitro exposure models. The order of the epithelial cell lines’ sensitivity to Ag was LA4 \u3e MLE12 \u3e C10. The macrophage sensitivity to Ag toxicity was C57BL/6 AM \u3e MARCO null AM, which indicated that the MARCO receptor was involved in uptake of the negatively charged Ag particles. These results support the idea that Ag nanosphere toxicity and NLRP3 inflammasome activation are determined by the rate of surface dissolution, which is based on relative surface area. This study highlights the importance of utilizing multiple models for in vitro studies to evaluate nanomaterials

Potential Role of the Inflammasome-Derived Inflammatory Cytokines in Pulmonary Fibrosis

Pulmonary fibrosis is a progressive, disabling disease with mortality rates that appear to be increasing in the western population, including the USA. There are over 140 known causes of pulmonary fibrosis as well as many unknown causes. Treatment options for this disease are limited due to poor understanding of the molecular mechanisms of the disease progression. However, recent progress in inflammasome research has greatly contributed to our understanding of its role in inflammation and fibrosis development. The inflammasome is a multiprotein complex that is an important component of both the innate and adaptive immune systems. Activation of proinflammatory cytokines following inflammasome assembly, such as IL-1β and IL-18, has been associated with development of PF. In addition, components of the inflammasome complex itself, such as the adaptor protein ASC have been associated with PF development. Recent evidence suggesting that the fibrotic process can be reversed via blockade of pathways associated with inflammasome activity may provide hope for future drug strategies. In this paper we will give an introduction to pulmonary fibrosis and its known causes. In addition, we will discuss the importance of the inflammasome in the development of pulmonary fibrosis as well as discuss potential future treatment options

Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MACRO

MARCO is the predominant scavenger receptor for recognition and binding of silica particles by alveolar macrophages (AM). Previously, it was shown that mice null for MARCO have a greater inflammatory response to silica, but the mechanism was not described. The aim of this study was to determine the relationship between MARCO and NLRP3 inflammasome activity. Silica increased NLRP3 inflammasome activation and release of the proinflammatory cytokine, IL-1b, to a greater extent in MARCO-/- AM compared to wild type (WT) AM. Furthermore, in MARCO-/- AM there was greater cathepsin B release from phagolysosomes, Caspase-1 activation, and acid sphingomyelinase activity compared toWT AM, supporting the critical role played by lysosomal membrane permeabilization (LMP) in triggering silica-induced inflammation.The difference in sensitivity to LMP appears to be in cholesterol recycling since increasing cholesterol in AM by treatment with U18666A decreased silica-induced NLRP3 inflammasome activation, and cells lacking MARCO were less able to sequester cholesterol following silica treatment. Taken together, these results demonstrate thatMARCO contributes to normal cholesterol uptake in macrophages; therefore, in the absence ofMARCO, macrophages are more susceptible to a greater inflammatory response by particulates known to cause NLRP3 inflammasome activation and the effect is due to increased LMP

Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MARCO

MARCO is the predominant scavenger receptor for recognition and binding of silica particles by alveolar macrophages (AM). Previously, it was shown that mice null for MARCO have a greater inflammatory response to silica, but the mechanism was not described. The aim of this study was to determine the relationship between MARCO and NLRP3 inflammasome activity. Silica increased NLRP3 inflammasome activation and release of the proinflammatory cytokine, IL-1β, to a greater extent in MARCO−/− AM compared to wild type (WT) AM. Furthermore, in MARCO−/− AM there was greater cathepsin B release from phagolysosomes, Caspase-1 activation, and acid sphingomyelinase activity compared to WT AM, supporting the critical role played by lysosomal membrane permeabilization (LMP) in triggering silica-induced inflammation. The difference in sensitivity to LMP appears to be in cholesterol recycling since increasing cholesterol in AM by treatment with U18666A decreased silica-induced NLRP3 inflammasome activation, and cells lacking MARCO were less able to sequester cholesterol following silica treatment. Taken together, these results demonstrate that MARCO contributes to normal cholesterol uptake in macrophages; therefore, in the absence of MARCO, macrophages are more susceptible to a greater inflammatory response by particulates known to cause NLRP3 inflammasome activation and the effect is due to increased LMP

Macrophage Fusion into Multinucleated Giant Cells In Vitro

Multinucleated giant cells (MGC) have been observed in a variety of granulomatous conditions, including microbial infections (e.g., tuberculosis), foreign body reactions to implants (e.g., medical devices), foreign body reactions to inhaled particles (e.g., engineered nanomaterials), and disorders of unknown etiology (e.g., sarcoidosis). Generally, MGC are morphologically classified based on the number and arrangement of nuclei. The two major types of MGC are foreign body giant cells and Langhans giant cells. These MGC are formed by the fusion of macrophages, often in response to persistent, foreign microorganisms or materials. Although MGC are known to be associated with granulomas, their involvement in the development of these conditions has not been well described. This is in part due to a lack of well-characterized models of MGC populations. The objective of this study is to develop an in vitro model of macrophage fusion in order to study MGC function. Previous reports have shown that MGC formation is induced by interleukin-4 (IL-4). Therefore, we investigated a model of IL-4-induced fusion in murine bone marrow-derived macrophages (BMdM). As expected, IL-4 treatment resulted in increased percent fusion of BMdM. The formation of MGC was optimized by modification of culture conditions, including alteration of the growth surface and treatment with either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Ongoing studies involve identification of molecules that regulate MGC formation. An increased understanding of this mechanism will provide additional targets to control fusion. Further development of this controlled in vitro model will facilitate future investigation of MGC inflammatory activity and contribution to pathogenesis of granulomas

Toxicity of Lunar and Martian Dust Simulants to Alveolar Macrophages Isolated from Human Volunteers

NASA is planning to build a habitat on the Moon and use the Moon as a stepping stone to Mars. JSC-1, an Arizona volcanic ash that has mineral properties similar to lunar soil, is used to produce lunar environments for instrument and equipment testing. NASA is concerned about potential health risks to workers exposed to these fine dusts in test facilities. The potential toxicity of JSC-1 and a Martian soil simulant (JSC-Mars-1, a Hawaiian volcanic ash) was evaluated using human alveolar macrophages (HAM) isolated from volunteers; titanium dioxide and quartz were used as reference dusts. This investigation is a prerequisite to studies of actual lunar dust. HAM were treated in vitro with these test dusts for 24 h; assays of cell viability and apoptosis showed that JSC-1 and TiO2 were comparable, and more toxic than saline control, but less toxic than quartz. HAM treated with JSC-1 or JSC-Mars 1 showed a dose-dependent increase in cytotoxicity. To elucidate the mechanism by which these dusts induce apoptosis, we investigated the involvement of the scavenger receptor (SR). Pretreatment of cells with polyinosinic acid, an SR blocker, significantly inhibited both apoptosis and necrosis. These results suggest HAM cytotoxicity may be initiated by interaction of the dust particles with SR. Besides being cytotoxic, silica is known to induce shifting of HAM phenotypes to an immune active status. The immunomodulatory effect of the simulants was investigated. Treatment of HAM with either simulant caused preferential damage to the suppressor macrophage subpopulation, leading to a net increase in the ratio of activator (RFD1+) to suppressor (RFD1+7+) macrophages, a result similar to treatment with silica. It is recommended that appropriate precautions be used to minimize exposure to these fine dusts in large-scale engineering applications