Towards Fish-Specific New Approach Methodolgies (NAMs) for Immunotoxicology: Evaluating the Cytokine Tumour Necrosis Factor Alpha (TNFα) as Reference Immunostimulant for Zebrafish In Vitro Studies
Abstract: Growing evidence suggests that many environmental pollutants can induce immunomodulatory and immunotoxic effects across species, including fish. Considering the critical role played by the immune system in the maintenance of health, the European Chemical Agency (ECHA) has recently classified immunotoxicity as a priority regulatory challenge. The effects of chemicals on the immune system of fish are typically characterised using in vivo experiments. However, scaling up these investigations for the 100,000+ chemicals currently on the market requires the urgent development of fish-specific new approach methodologies (NAMs).
In human immunomodulation and immunotoxicity testing, immunostimulants - such as the pleiotropic cytokine tumour necrosis factor (TNFα) - are commonly used to assess the effects of chemicals on activated immune cells. Here, we hypothesise that TNFα can be considered as the reference immunostimulant for fish in vitro immunology studies as it is for human studies. Yet, the effects of TNFα on fish cell lines are largely unexplored, especially in the context of NAMs. Given the lack of fish immune cell lines and the widespread expression of cytokine receptors in non-immune cells, we also hypothesised that ZF4 cells can be used as a proxy to detect the immunomodulatory activity of both endogenous and exogenous substances. To address this knowledge gap, we combined traditional ELISA assays with high-content methodologies to perform an in-depth characterisation of the functional (i.e. interleukin 6 release), morphological, and transcriptomic responses of zebrafish fibroblast-like ZF4 cells to TNFα.
Our results indicated that ZF4 cells are highly responsive to TNFα, mimicking the response of human cells. Exposure to 10, 25, 50, and 100 ng TNFα/mL induced a rapid concentration-dependent increase in IL-6 secretion over a 24-hour period, with a peak secretion between 15 and 30 minutes. These results were used to design further studies aimed at characterising short-term (15-30 minutes) and longer-term (24 hours) transcriptomic effects via RNAseq and morphological effects via high-content cell imaging approaches. Our findings suggest that exposure to TNFα at 50 ng/mL can provide a robust method for immunostimulation in non-immune zebrafish cells, supporting its application in fish-specific NAMs for environmental toxicology.
Disclaimer/ Disclosure (optional): This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) iCASE studentship co-funded by AstraZeneca under the London Interdisciplinary Doctoral Programme (LIDo) (Ref. 2879914).
Declaration of competing interest: GH was in receipt of a scholarship (awarded to LM-C) co-funded by AstraZeneca. SFO is an employee and share holder of AstraZeneca, a biopharmaceutical company with an interest in the discovery, development and commercialisation of prescription medicines.
Author(s):
Gian Hobbs King's College London United Kingdom This Author Is the Presenter
Stewart F. Owen AstraZeneca United Kingdom
Christer Hogstrand King's College London United Kingdom
Luigi Margiotta-Casaluci King's College London United Kingdom
Towards Fish-Specific New Approach Methodolgies (NAMs) for Immunotoxicology: Evaluating the Cytokine Tumour Necrosis Factor Alpha (TNFα) as Reference Immunostimulant for Zebrafish In Vitro Studies
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Poster & Poster Corner Discussion