Effects of subtoxic concentrations of TiO 2 and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production Britta Andersson-Willman a , Ulf Gehrmann a , Zekiye Cansu a , Tina Buerki-Thurnherr b , Harald F. Krug b , Susanne Gabrielsson a , Annika Scheynius a, ⁎ a Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden b Laboratory for Materials — Biology Interactions, Swiss Federal Laboratories of Materials Testing and Research, St. Gallen, Switzerland abstract article info Article history: Received 18 April 2012 Revised 21 June 2012 Accepted 17 July 2012 Available online 27 July 2012 Keywords: Nanoparticles Human peripheral blood cells Dendritic cells Natural killer cells Exosomes Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investi- gate if commercially available TiO 2 and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO 2 or ZnO nanoparticles at concentrations from 1 to 100 μg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO 2 nanoparticles. Non-toxic exposure, 10 μg/mL, to TiO 2 and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO 2 nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cul- tures. TiO 2 or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO 2 and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions. © 2012 Elsevier Inc. All rights reserved. Introduction Nanoparticles have at least one dimension in the nanometer (nm) range and are comparable to the size range of small biological entities. Advanced engineered materials possess special properties including a large surface area to volume ratio, whereby they take on novel proper- ties compared to those seen in the bulk scale. Metal oxide nanoparticles have been extensively studied from a toxicology point of view (Schilling et al., 2010) and many studies have focused on titanium dioxide (TiO 2 ) and zinc oxide (ZnO) nanoparticles due to their frequent use in sun- screens and cosmetics (Nohynek et al., 2008; Schilling et al., 2010). As stated in a recent review, TiO 2 and ZnO nanoparticles do not penetrate the skin and thus they have been claimed to not pose a potential risk to human health when applied onto undamaged skin (Nohynek et al., 2010). However, due to semiconductor properties of ZnO nanoparticles, the industrial and electronic applications have gained much attention with other exposure routes and risks at hand. The inflammatory effects of TiO 2 and ZnO nanoparticles have been addressed both in vitro (Heng et al., 2011; Winter et al., 2011) and in vivo (Cho et al., 2012; Monteiro-Riviere et al., 2011) but we are far from understanding how nanoparticles affect the human immune system. Most knowledge is derived from studies on cell lines that are reproducible and well standardized assays. However, it is unclear how well these results translate to primary human cells. Only a few studies have tested the effect of nanoparticles on human peripheral blood mononuclear cells (PBMC) (Boraschi et al., 2012; Boscolo et al., 2010; Greulich et al., 2011). The use of PBMC allows for the simul- taneous analyses on the effects of nanoparticles on several different important immune cells such as T cells, B cells, monocytes and natural killer (NK) cells. Thus, exposure of PBMC to silver nanoparticles showed specific effects on monocytes while T cell proliferation remained unaffected (Greulich et al., 2011). Dendritic cells (DC) are the antigen presenting cells (APC) in the body with the highest capacity to initiate and direct immune responses and most likely to encounter and take up nanoparticles in vivo. Since they are so few in peripheral blood, less than 1%, an established method is to generate monocyte derived den- dritic cells (MDDC) for in vitro analyses (Vallhov et al., 2007). To exert their function, immune cells need to communicate. One way is through production and release of exosomes. Exosomes are Toxicology and Applied Pharmacology 264 (2012) 94–103 ⁎ Corresponding author at: Karolinska Institutet, Department of Medicine Solna, Trans- lational Immunology Unit L2:04, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden. Fax: +46 8 335724. E-mail address: annika.scheynius@ki.se (A. Scheynius). 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2012.07.021 Contents lists available at SciVerse ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap