control. Discussion Advances in the nanosciences over the past several decades have led to engineering nanomaterials with extremely precise physicochemical properties (e.g. ENM in consumer and medical products, in two key innate immune cell models, e.g. RAW 264.7 cells (macrophages) and differentiated MPRO 2.1 cells (promyelocytes/neutrophils). The results showed that despite a generation of reactive oxygen species, exposure to 20 nm citrate-coated AgNP was not associated with major oxidative damage, inflammatory responses, nor cytotoxicity. Nevertheless, and most importantly, pre-exposure to the AgNP for 24 h enhanced RAW 264.7 cell phagocytic ability as well as the release of inflammatory cytokine interleukin-6 in response to lipopolysaccharide (LPS). In MPRO 2.1 cells, AgNP pre-exposure also resulted in enhanced phagocytic ability; however, these cells manifest reduced cell degranulation (elastase release) and oxidative burst in response to phorbol myristate acetate (PMA). Taken together, these findings indicated to us that exposure to AgNP, despite not being directly (cyto)toxic to these cells, had the potential to alter immune cell responses. The findings underscore the import of assessing immune cell function post-exposure to ENM beyond the standard endpoints such as oxidative stress and cytotoxicity. In addition, these findings further illustrate the importance of understanding the underlying molecular mechanisms of ENM-cellular interactions, particularly in the immune system. exposure of rats to 20 nm AgNP (intravenously, daily for 28 days) led to increased spleen weight and neutrophil infiltration reduced thymic weights and natural killer (NK) cell Rabbit Polyclonal to TOP1 activity, and suppression of T-cell dependent antibody production. Those findings suggested that despite a lack of direct tissue or system toxicity, chronic exposure to certain AgNP could be associated with adverse health outcomes. Previous research has exhibited direct cytotoxicity of ENM (including some AgNP) on several immune cell models (Yang et al. 2012; Hamilton et al. 2014; Aldossari et al. 2015; Liz et al. 2015; Alsaleh et al. 2016; Vallieres et al. 2016; Muller et al. 2018). Furthermore, emerging evidence has suggested potential immunomodulatory properties of metal and metal-oxide ENM at sub-cytotoxic concentrations which do not result in reduced cell SIB 1757 viability (Comfort et al. 2011; Andersson-Willman et al. 2012; Seydoux et al. 2014). Nevertheless, due to differences in AgNP physicochemical properties, choices of cellular models, variations in toxicological/immunological endpoints examined, comparisons between previous studies are almost impossible. Indeed, despite efforts, this has been a challenge in the basic assessment of AgNP safety (Bonner et al. 2013; Xia SIB 1757 et al. 2013). Accordingly, this study investigated cellular responses to AgNP in two key innate effector immune cell models, i.e. a macrophage model (RAW 264.7 cells) and a promyelocyte/neutrophil model (MPRO 2.1 cells). Specifically, the studies here assessed direct cellular toxicities in response to 20 nm AgNP; endpoints evaluated in the cell lines included viability, AgNP uptake, reactive oxygen species (ROS) generation, oxidative stress, and inflammatory responses. These studies also investigated potential changes in cellular function and activation to known immunological stimulants (i.e. 24 h post-exposure to the test AgNP). Materials and methods Nanoparticle characterization Hydrodynamic size (nm), zeta () potential (mV), and polydispersity index (PDI) were measured for 20 nm BioPure? citrate-coated AgNP (NanoComposix, San Diego, CA) using a Zetasizer (Malvern, Westborough, MA) in DI water (nanoparticle vehicle) and cell culture media (Table 1). Transmission electron microscopy (TEM) was used to confirm the size and shape of AgNP (Supplementary Physique S1). Table 1. Characterization of test AgNP in DI water and cell culture media. for 5 min and media was removed and replaced with phenol SIB 1757 red-free DMEM/F12 HyClone media (GE, Pittsburgh, PA). Thereafter, MTS reagent was added to each well and the plates were incubated at 37 C for 20 min until color development. The plates were then centrifuged at 300 for 5 min and supernatants from each were collected and transferred to new 96-well plates. Absorbance in each well was then measured at 490 nm in a Synergy HT system (BioTek, Winooski, VT). Cells that were treated with hydrogen peroxide (H2O2; 10 mM) for 60 min was used as a positive control. Nanoparticle uptake by cells AgNP uptake was measured by inductively coupled plasma mass spectrometry (ICP-MS) as described previously (Aldossari et al. 2015). In brief, cells were seeded into 24-well culture plates and produced to 80% confluency. A concentration of 50 g/ml was utilized for most subsequent experiments because it was not associated with reduced viability and is more relevant to previous literature than would be the higher doses examined in the viability studies. Following five washes with ice-cold phosphate-buffered saline (PBS, pH 7.4) to remove any suspended AgNP that had not been internalized by cells, the cells were.