Immunotoxicity and Cellular Stress

By BioNanoNet

Nanomaterials can activate or suppress the immune system, there are many ways in which nanomaterials may drive these immune responses, through the generation of potentially damaging substances such as reactive oxygen species, or by exerting direct mechanical stress on cellular membranes or interactions with DNA, but also through indirect effects, where induction of chemical messages from other cells types leads to pro-inflammatory conditions and activated immune cells. Effects may be general or restricted to specific conditions, for example an ongoing cell activation leading to a diseased state. The modulatory effects of nanomaterials is highly variable, and therefore no single endpoint is solely suitable when evaluating the immunosafety of nanomaterials, and, at this time, there are no standardized protocols available that provide inclusive assessment of nanomaterial toxicological issues, which would allow the inference of relative risk evaluations, and a reliable risk assessment. Hence, robust, cheap and reproducible tests which provide information on a number of relevant endpoints are developed, to give a clear account of immune modulation. When nanomaterials enter the body, they will interact with a combination of numerous cell systems, whether it be the constituents of the respiratory tract, or that of the immune system. Numerous defense mechanisms may be elicited, including pro-inflammatory conditions and antioxidant defenses, which may lead to cellular stress, acute and chronic inflammation, genotoxicity, and potentially carcinogenesis. To aid our research and to provide robust, cheap and reproducible assays, a panel of stable human reporter cell lines was established which express Luciferase, GFP, or RFP, under the control of selected promoters. These can be used to evaluate effects of chemicals and nanoparticles on cell stress induction and expression of inflammatory cytokines and chemokines, to determine the molecular pathway that has been activated, and to assess the method of cellular stress that has occurred. Luciferase, GFP, and RFP as a reporter genes are highly sensitive and easy to test using a standard plate reader with a capacity for luminescence and fluorescence measurements. Reporter assays are suitable for medium-to-high throughput formats. Positive results from screening assays are confirmed using various methods to detect gene transcripts and proteins both in cell lines and in relevant primary cells, in particular lung epithelial cells, monocytes/macrophages and T-cells. Our experiences show that most suspected effects detected in screening are confirmed by direct testing of primary human cells. List of available assays: Cytotoxicity and Proliferation: Alamar Blue®, Toxilight®, CytoTox 96®, Cell Titer Blue®, MTT, ³[H]-Thymidine. Luc reporter cell lines: In A549 human lung epitelial cells IL-6, IL-8, TNF-α, NF-κB. In Jurkat human T cells IL-4, IL-6, IL-8, IL-13, IFN-γ, TGF-ß, TNF-α, NF-κB. In HeLa IL-8. In HEK Eotaxin-1. GFP cell line: In A549 IL-8, other proi