Quantum dots trigger immunomodulation of the NFκB pathway in human skin cells
Introduction
Quantum dots (QDs) are crystalline semiconductors approximately 1–20 nm in diameter. QD nanocrystals composed of CdSe cores and ZnS shells have received attention due to their unique electronic and optoelectronic properties at nanoscale levels and their widespread applications (Azzazy et al., 2007, Delehanty et al., 2008, Hild et al., 2008, Medintz et al., 2008, Michalet et al., 2005, Jamieson et al., 2007, Li et al., 2007, Walling et al., 2009). Because of their unique characteristics, QDs are used at increasing rates for a wide variety of industrial and consumer-based applications, including biomedical imaging agents, inks, and solar panels (Michalet et al., 2005, Alivisatos et al., 2005, Gao et al., 2004, Roco, 2003). QDs may also pose risks to human health, where unintended exposure to nanomaterials may occur at the workplace or during end product use via inhalation, dermal absorption, or gastrointestinal tract absorption (Rzigalinski and Strobl, 2009). Dermal exposures to QD particles have shown toxicities due to heavy metal exposure and/or the production of reactive oxygen intermediates (ROIs) (Ryman-Rasmussen et al., 2007, Kirchner et al., 2005, Derfus et al., 2004).
Due to the growing number of potential uses that are offered by QD materials, consumer handling and manufacturer exposure to QDs is likely to increase. Nanoscale materials are thought to impose increased adverse effects on organisms than microscale materials because of their finer sizes and corresponding larger specific surface areas per unit mass (Monteiller et al., 2007, Maynard and Kuempel, 2005, Oberdorster et al., 2005). However, it is largely unknown which specific pathways or subcellular mechanisms of action are triggered as a result of QD exposure. Many investigators have shown that QDs can be internalized into cells and others have speculated the route of entry for particular QDs (Zhang and Monteiro-Riviere, 2009, Duan and Nie, 2007, Jaiswal et al., 2003), but what are the mechanisms of injury and key participants on the molecular level in the cell and how do those processes develop? We hypothesized that immune mediators of inflammation may be initiated in the exposed cell layers.
In an attempt to fill this gap, human epithelial keratinocytes (HEK), found in the epidermis, and human dermal fibroblasts (HDF), located in the dermis, were utilized to query the molecular interactions with QDs. Dermal cells were chosen because contact with the skin is one of the routes of exposure to QDs. Zhang and Monteiro-Riviere (2008) and Mortensen et al. (2008) both concluded that QDs of similar or identical structure and composition to those used in this study could penetrate through the epidermis into the dermis, especially with flexing of the skin or by way of hair follicles. Microscopy from their publications revealed that a considerable portion of the dose penetrated to the dermis. These studies used concentrations which ranged from 1 to 2 μM. However, this manuscript focuses on the nanomolar dosing concentration range which resulted in a less toxic response, yielding cellular viability between 15% and 100% in both HEK and HDF cells. A recent development in the nanotoxicology literature noted the importance of conducting in vitro experiments with a concentration range of nanoparticles that would not fully overwhelm the culture, as this would likely be inconsistent with equivalently dosed in vivo studies (Oberdorster, 2010). Similarly, Zhang et al. (2006) found genetic perturbation in dermal cells exposed to 8 and 80 nM concentrations of silica-coated QDs. From this range, we selected the lowest observable adverse effect level (LOAEL) concentrations of 30 and 60 nM to further investigate the mechanisms of cellular pathway stimulation by probing for adverse or protective responses in the fibroblasts. The human dermal fibroblast cell line was of interest partially due to its proximity to the vascular system and its importance in maintaining the structural framework of the tissue (Fig. 1). Also, fibroblasts possess an elaborate cytokine response system, which allows these sentinels to initiate the process of inflammation (Le et al., 1987).
In an effort to increase current knowledge regarding pathways of the human cellular response to QDs, we have quantitatively investigated effects of an engineered QD on the expression of 50 unique genes in HDF cell cultures. In this study, we compared the dose-response and time-course effects of CdSe/ZnS-COOH QD nanoparticles in cells that induce or suppress one or more of the following effects: inflammation, immunoregulation, apoptosis, and cellular stress. Specifically, we found that many mRNA perturbations occurred in genes of the NFκB pathway, which is involved in each of these processes. NFκB is a major transcription factor responsible for regulating genes of both the innate and adaptive immune response (Livolsi et al., 2001). NFκB becomes activated through distinct signaling components. Inactivated, cytosolic NFκB is complexed with the inhibitory IκBα (NFKBIA) protein. A variety of extracellular signals can be stimulated via integral membrane receptors, which can then activate the enzyme IκB kinase (IKK or IKBKB). The role of IKK is to phosphorylate the NFκB-associated IκBα protein, resulting in ubiquination and dissociation of IκBα from NFκB. IκBα is degraded by the proteosome and the liberated NFκB is then translocated into the nucleus where it binds to specific DNA motifs in promoters, termed response elements. Here, it can upregulate genes involved in immune cell development, maturation, and proliferation, as well as those dedicated to survival, inflammation, and lymphoproliferation (Zandi, 1997). Conversely, a suppression of nuclear NFκB can result in TNFα-induced apoptosis (Beg and Baltimore, 1996, Liu et al., 1996, Van Antwerp et al., 1996, Wang et al., 1996). This decrease in nuclear translocation is due to increased levels of IκBα, which we found to be upregulated in our study.
Quantitative-PCR revealed both time and concentration dependent patterns of gene regulation. From our analyses of these data, we deduced that the particles used in this study influenced regulation of genes and proteins along the NFκB pathway, as evidenced by deviations of relevant gene expression (NFκB, IL-1B, IRAK1/2, CASP1). Results from western blotting also revealed increased induction of inflammatory proteins (HMOX-1, IL-1B, TNF-α) caused by stress, which is thought to arise from ROI and/or metal-induced toxicity in response to QD exposure.
To our knowledge, this work is the first to examine immune and inflammatory responses arising from QD exposure in dermal cells. Very few studies exist that have analyzed this type of cellular response to QDs. Hoshino et al. (2009) found that direct injections of QD/nucleotide complexes into the peritoneal cavity of mice resulted in inflammation with the infiltration of inflammatory cells. They also found that the same complex induced the production of both proinflammatory cytokines and chemokines. Rehberg et al. (2010) recently found that, depending on surface modification, QDs can modulate leukocyte adhesion and migration. Since such findings of cellular perturbation have been presented in the literature, further inquiry of the mechanisms of gene induction or suppression is necessary.
Section snippets
Quantum dot nanocrystal characterization
CdSe/ZnS-COOH crystalline quantum dots were purchased from Ocean Nanotech, LLC (Springdale, Arkansas). Characterization data is summarized in Table 1. Particle size and zeta potential were characterized in-house via Malvern ZetaSizer Nano ZS (Malvern Corp., Worchestershire, UK). Particle characterization was performed on the particles while in Milli-Q ultrapure water. Chemical composition was determined via inductively couple plasma-mass spectroscopy (Elan DRC II, PerkinElmer SCIEX); no metal
Cellular morphology
HDF and HEK cells were exposed to a range of CdSe/ZnS QDs from 1 nM to 120 nM for 8 and 48 h (Fig. 2). Phase and fluorescence images revealed an internal accumulation of QDs, which increased with both time and concentration. As a result of these exposures, HEK cell density decreased more severely than HDF cell density (Supplemental Figures 2 and 3). The decreasing number of HEK cells corresponds to the amount of detectable fluorescence at the plane of the cells. Few detectable morphological
Discussion
QD particles modulated gene and protein expression relating to oxidative stress, apoptosis, inflammation, and non-inflammatory immune response pathways, especially genes typically included in the NFκB pathway. Similar to previously published high exposure concentration studies with nanoparticles, we found that the observed low exposure concentration effects were dependent on QD dosing concentration and exposure time (Kirchner et al., 2005, Hoshino et al., 2004, Prasad et al., 2010, Delehanty et
Acknowledgments
AAR and CMS thank Dr. Christine Heaps, Texas A&M University, for generously donating the porcine skin used in Fig. 1. AAR and CMS thank Drs. Stephen Safe and Yanin Tian for the use of Lamin and p65 NFκB antibodies, respectively. CMS thanks the Department of Physiology & Pharmacology and the DuPont Company for financial support. MFC thanks the Department of Veterinary Pathobiology and NIH NIAID AI073888 for financial support. II thanks the Department of Veterinary Physiology & Pharmacology.
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