Characterization of immunomodulatory effects of nanoparticles in vitro

Nanoparticles are ultrafine particles at a size within the low nanometer range (1-100nm) and represent an intermediate position between bulk materials and molecular structures. In the last decade there has been a strong increase in the use of engineered nanoparticles, but despite their growing presence and their exposure to humans still little is known about their risks for human health. Their interaction with biological systems is relatively unknown and therefore it is necessary to detect their capacity to interfere with vital biochemical processes and harm human health. Within the scope of this project we address the interaction of nanoparticles with the human immune system. During activation of the cellular immune system, Th1-type cytokine interferon- (IFN-) induces enzyme GTP- cyclohydrolase I, which gives rise to neopterin production by activated human macrophages. In parallel, IFN- induces enzyme indoleamine- 2,3-dioxygenase (IDO) which converts tryptophan to kynurenine. The relevance of these pathways in human diseases is indicated by the fact that tryptophan degradation and neopterin formation are increased during several disease states involving Th1-type immune activation and are of significant predictive value regarding outcome of patients. Applying peripheral blood mononuclear cells (PBMC) freshly isolated form healthy donors and THP-1 cells our group has established a sensitive in vitro system to detect immunomodulatory properties of drugs and compounds in vitro. In supernatants of PBMC stimulated with mitogens such as phytohaemagglutinin (PHA) or concanavalin A (Con A) and THP-1 cells stimulated with lipopolysaccharide (LPS), the increase of neopterin concentrations (ELISA) and tryptophan degradation (HPLC) will be measured. The influence of nanomaterials TiO2 , ZnO, Al2 O3 , CeO 2 and carbon nanotubes will be examined in stimulated and unstimulated cells. Results will be compared to interferon- (IFN-) levels. In addition, capacity of nanomaterials to induce or reduce intracellular oxidative stress will be performed in THP-1 cells. Further, nuclear factor kappa B (NF-B) activation will be measured in LPS-stimulated and unstimulated myelomonocytic THP-1 expressing a NF-B inducible reporter system. Effects of nanoparticles on cell viability will be tested to detect possible toxic effects on the cells. Well defined and stable solutions of nanoparticles, e.g. TiO2 , will be obtained from Dr. Nathalie Herlin-Boime, SPAM-LFP, French Atomic Energy Commission in Gif sur Yvette in France within the nanoLINEN consortium established in the ERANET initiative.

The aim of this project was the identification of immunomodulating properties of nanoparticles in different cell systems in vitro. Due to the increasing applications of nanoparticles in consumable products and the lack of in vitro data concerning central T-helper immune biomarkers, this project represents a great advancement for a better risk-benefit assessment of wide spread and commonly used nanoparticles. The goal of the project was the testing of different particle sizes concerning their size-dependent effects on immunologic signaling cascades. Such validation can help to get a better insight to validate whether the immunomodulating effect of the particle is due to the small size (nano) or derives from the chemical itself independent from its size. The negative rumored effect of nanoparticles is due to their small size and huge increase of the total surface area, which results in highly reactive particles. Nevertheless our results could not show a relevant size dependent effect, and the size-dependent toxicity of the particles could not be confirmed.For this purpose several commonly used nanoparticles as zinc oxide, elementary silver, aluminium oxide, carbon nanotubes and titanium dioxide have been tested regarding their influence on inflammatory signaling pathways. With the help of different measurements which are able to detect the activation of the central inflammation marker neopterin and the degradation of the essential amino acid tryptophan, the influence of different nanoparticles concerning their pro- or anti-inflammatory properties can be monitored. Furthermore, expression of NF-kB, a central inflammatory activation marker, and the detection of the amount of reactive oxygen species can provide a hint for their mode of action regarding immunomodulation. For the most part of the tested particles we observed anti-inflammatory effects, which could be shown by an inhibition of neopterin production and a rescue in tryptophan degradation after previously generated inflammatory milieu. Interestingly, treatment with different zinc oxide particle sizes resulted in an inhibition of inflammatory signaling cascades. Particles of all sizes exerted similar results and could confirm the known anti-inflammatory property, however could not confirm a size-dependent effect. Additionally all particles have been tested for their ability to neutralize or generate reactive oxygen species (ROS) in lung epithelial cells. Interestingly, zinc oxide and carbon nanotubes increased the amount of free radicals, and instead aluminium oxide and silver inhibited and neutralized the generated ROS. Those differentially observed results agree well with the different outcomes in different cell types. Thus, it is of utmost importance to test the influences of nanoparticle exposure on different cell types to receive interactive cell communications and cell responses.