We will develop methods using existing in vitro neutrophil and transgenic zebrafish (ZF) embryo models to assess the initiation, progression and resolution of inflammation. The alternative models will be applied to test the hazard potential of nanomaterials (NMs) of varied physicochemical properties, focusing on NMs for which in vivo rodent pulmonary data already exists in order to assess the relevance and limitations of the alternative models.
HL60 neutrophil-like cell line and primary neutrophils (isolated from human blood) will be exposed to NMs (copper oxide, carbon nanotubes, silver, zinc oxide, organic pigments) of varied physicochemical properties (e.g. composition, size and shape). NM cytotoxicity will be ranked and sub-lethal concentrations identified to investigate the mechanism underlying NM mediated neutrophil activation by NMs including; intracellular signaling via calcium and ROS, oxidative stress, cytokine production, respiratory burst and phagocytic function, as well as cell death including netosis and apoptosis. Research using ZF embryos will be conducted in UK Home Office approved facilities using non-protected early life history stages (age<120h post-fertilisation). Neutrophil responses to NMs will be visualised and quantified over time in transgenic ZF embryos (with fluorescent neutrophils) following micoinjection, at a range of doses. The mechanism underlying neutrophil activation and resolution in ZF embryos will be investigated via assessment of cytokine (e.g. CXCL8) and antioxidant expression, ROS production, calmodulin expression and neutrophil apoptosis. A range of transgenic ZF embryos are available to further probe the involvement of specific cellular and molecular events in the activation and resolution of NM inflammatory responses (e.g. CXCL8 knockout). Data obtained will be compared to existing in vivo data using in vitro-in vivo extrapolation (IVIVE), to explore the relevance of the alternative models.
Verdon R et al. (2020) Neutrophil activation by nanomaterials in vitro: comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line. Nanotoxicology, in press. doi: 10.1080/17435390.2020.1834635
Johnston HJ et al. (2018). Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials. Critical Reviews in Toxicology 48(3):252-271. doi: 10.1080/10408444.2017.1404965