Project Title: Defining an evolutionary conserved NP-associated Adverse Outcome Pathway (NP) for immune cell cytotoxicity

 

Objectives: NP may enter in immune cells through hijacking the endocytic system that is a central component of the innate immune response[1]. We will exploit primary cultures of immune cells to visualise the cellular uptake, cytotoxicity and innate immune response upon NP exposure. Through adopting a systems toxicological approach we will temporally resolve the pathway by which the NP exert toxicity on the immune cells of the earthworm Eisenia fetida and woodlouse Porcellio scaber. Immune cells will be isolated from the coelomic cavity and heamocil, respectively, and maintained in culture for 24-72 h. These cells represent a spectrum of immune cell types from amoebocytes to granulocytes. We will study the uptake and modification of Ag and Zn NP to establish their specific entry pathway, and determine the chemical environment that causes compound dissolution and subsequent chemical transitioning. We will exploit Coherent anti-Stokes Raman spectroscopy (CARS-RAMAN) to visualise the real-time uptake of NP (Figure).

After NP internalisation the chemical environment changes, with a quick lowering of the compartment pH that may result in the dissolution of metal NP and ion release. Subsequent to NP dissolution, the metal ions may be released into the cytosol circumventing normal plasma membrane flux control. We will use high content (HC) temporally resolved transcriptome analysis to define the adverse outcome pathways (AOP) linking the internal release of ions to cytotoxicity. Prediction of the consequence of internal ion release will be based on cytotoxic endpoints (genotoxicity/chromatin effects, ROS generation, cellular detachment and associated phosphorylation cascades). We will interrogate the response networks to identify the key nodes in the response pathways and mechanistically validate these predictions.

 

Expected Results: 1. establishing the key NP parameters that influence uptake, by characterising the relationship between NP size and coating with the internalisation pathway; 2. determining kinetics of NP dissolution by temporally tracking changes in the endocytic compartments and effects on the internalised parent NP; 3. identifying the molecular event initiating AOP (how NP transmits its toxicity and compromises the immune system), by temporally resolved transcriptomic analysis.

[1] Kunzmann A. et al. (2011). Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation. Biochim. Biophys. Acta 1810: 361.