Summary

A number of industrialized countries, including Canada, have opted for the development of nanotechnologies given their immense economic potential. Québec is already home to many companies that produce nanoparticles (NP), and the establishment of others can be expected in the near future. This means that the number of workers employed in nanotechnology fields in general or NP manufacture and synthesis will increase significantly in the coming years. Preventing the development of NP-related occupational diseases and work injuries, which are often of inflammatory in origin, is thus crucial. A growing body of scientific data demonstrates the toxic, adverse effects of some NPs, but we do not know either their toxicity or the effectiveness of measures to contain them. Inflammation is among the toxic effects of NPs most commonly reported in the scientific literature. In vivo studies in which NPs are administered to rodents by inhalation (the most likely NP exposure route for workers) report elevated counts of neutrophil immune cells, the key orchestrators of inflammation. Curiously, despite this, there is very little data on direct interactions between NPs and neutrophils. As NPs can reach the bloodstream not only through inhalation but also through ingestion or skin absorption, the idea that neutrophils, the most abundant type of white blood cell in human blood, would interact with NPs is plausible.

This project was meant to fill this gap. The first objective was thus to demonstrate the effect of NPs on the physiology and functions of the human neutrophil (in vitro study). The second objective (in vivo study) was to assess the effect of NPs on the inflammatory response in mice.

For the in vitro study, freshly isolated neutrophils from consenting healthy donors were treated with a specific NP to assess modulatory capacities on several functions related to the inflammatory process. For the in vivo study, the murine air-pouch model was used. Briefly, an air pouch was produced by subcutaneous injection of sterile air and an NP in a variety of concentrations was administered for diverse periods of time (parameters selected based on the in vitro results). The cells colonizing the pouch were then counted and identified to assess neutrophil infiltration.

By using an experimental approach that included in vitro as well as in vivo investigation, we were able to determine which NPs affect neutrophil physiology the most and which provoke neutrophil infiltration in vivo. For example, in vitro, CeO₂, TiO₂ and ZnO trigger differential effects on neutrophil phagocytosis and degranulation, and silver (Ag) NPs, depending on their diameter,  cause atypical neutrophil death. ZnO and TiO₂ activate neutrophils and delay their apoptosis. In vivo, we noted that TiO₂ and dendrimers trigger neutrophil infiltration, whereas other NPs, such as fullerenol (C₆₀(OH)_n), do not.

In sum, our results demonstrate that strict classification of NPs according to their proinflammatory potential is difficult and that a better approach is to give a more nuanced picture in which the effects of a particular NP on the physiology of human neutrophils in vitro and on the capacity to induce neutrophil infiltration in vivo are taken into consideration. Incidentally, the impacts of NPs are extremely varied. It has been demonstrated that NPs do not all act in the same way in vitro and in vivo and that they can have the completely opposite effects depending on their initial diameter.

The results of this project carried out with isolated cells from healthy individuals will make it possible to use cells from workers who may have been exposed to NPs to study functions of neutrophils that might have exaggerated functional responses because they were already preactivated, a well-known concept in inflammation. Contrariwise, if, for example, the phagocytosis function of the neutrophils of these workers turns out to be diminished, then the  workers may be more susceptible to infection, as the purpose of this neutrophil function is to ingest and destroy pathogens. These observations, together with those described by other teams studying other facets of NP toxicity, will help preventionists in making risk management decisions regarding occupational exposure to NPs.