Soluble silver ions from silver nanoparticles induce a polarised secretion of interleukin-8 in differentiated Caco-2 cells
Introduction
With the advances of nanotechnology, manufactured nanoparticles came on the market with promises of improved optical, catalytical, electronical or antimicrobial properties (Vigneshwaran et al., 2007; El-Nour et al., 2010; León-Silva et al., 2016; Syafiuddin et al., 2017). However, their smaller size and higher reactivity could also raise their toxicity. Among them, silver nanoparticles (AgNPs) are increasingly used in consumer products because of their antimicrobial properties (Wijnhoven et al., 2009). According to the Project of Emerging Nanotechnologies (The project on emerging nanotechnologies, 2019), AgNPs are found in the highest number of consumer products containing nanoparticles, with 50 % of the consumer products containing silver (The project on emerging nanotechnologies, 2019). In particular, "health and fitness" and "food and beverages" applications are the most represented categories for AgNPs, potentially resulting in their ingestion (Laloux et al., 2017). The presence of AgNPs has also been proven in the food additive E174 added in pastry decorations such as chocolates or silver pearls (Verleysen et al., 2015). Aside this example, AgNPs are not directly incorporated in food but can migrate into it from packaging materials, cooking instruments, cleaning sprays or storage boxes (Wijnhoven et al., 2009; Emamifar et al., 2012; Cushen et al., 2014; Echegoyen and Nerín, 2013; Hauri and Niece, 2011; Song et al., 2011; von Goetz et al., 2013). Even if the potential migrated amount remains low, the increasing use of AgNPs in consumer products could dramatically raise the consumer exposure to AgNPs. Moreover, AgNPs constitute some unauthorised food supplements whose consumption can lead to up to 0.02 mg/kg BW/day exposure (Larsen et al., 2015). After ingestion, these AgNPs will come in contact with the gut. Although the cytotoxicity of AgNPs has been largely studied, their effect on inflammation remains controversial.
Because of its major role in inflammation, we decided to focus on the production of interleukin-8 (IL-8) by intestinal epithelial cells (IECs), a chemokine involved in inflammatory processes. For this purpose, Caco-2 cells were used as an in vitro model of the gut mucosal barrier. Although isolated from a colonic adenocarcinoma (Fogh and Trempe, 1975), they differentiate spontaneously in cells presenting characteristics similar to small intestine enterocytes such as the presence of active tight junctions and efflux pumps (Artursson, 1990; Sambuy et al., 2005; Smetanová et al., 2011). These cells are thus commonly used as a simple in vitro model of the small intestine in pharmaco-toxicology studies. In particular, they are the most widely used for nanomaterial translocation assessment (Braakhuis et al., 2015; Georgantzopoulou et al., 2015).
Although toxicity of AgNPs has been extensively reported in the literature, the involvement of silver ions (Ag+) in their mode of action still remains unclear. These ions, as a subproduct of Ag0 oxidation (Reidy et al., 2013), are commonly found in AgNPs suspensions in variable proportions that can go even up to 69 % of total silver content (Beer et al., 2012), depending on the synthesis method (Beer et al., 2012), the AgNPs size (Bouwmeester et al., 2011) and concentration (Hadioui et al., 2013) or the chemical nature of their coating (van der Zande et al., 2012). Together with different chloride complexes (Behra et al., 2013), Ag + forms the "soluble Ag fraction" in AgNPs suspensions. Although silver salts are known to be toxic (Miura and Shinohara, 2009), only a few studies have addressed this issue. Most of the published reports concerning AgNPs have not distinguished the effect of soluble Ag from the global observed effects (Zhao and Wang, 2012). Papers addressing this issue have generally compared the effect of a silver salt such as acetate or nitrate as a source of Ag+ (Böhmert et al., 2015; Bilberg et al., 2011; Martirosyan et al., 2016; Navarro et al., 2008; Yin et al., 2011). Only a few tests have been performed with the soluble fraction separated from AgNPs suspensions although this was recommended by Beer et al. (2012) for all studies concerning metallic nanoparticles such as silver or copper (Beer et al., 2012). In addition, AgNPs toxicity was suppressed by cysteine, which inactivates soluble Ag by complexing the ions (Zhao and Wang, 2012). Another way to assess the involvement of this soluble Ag is to separate it from the suspension, either by ultracentrifugation (Beer et al., 2012) or by filtration through a membrane with an appropriate cut-off (Zhao and Wang, 2012). Silver ions could also be separated from the rest of the suspension by ion exchange resin (Hadioui et al., 2013) although this is less used in literature.
The gut is a major immune organ, with about 60 % of the total immunoglobulin content of the human body (Salminen et al., 1998). Within this organ, the gut-associated lymphoid tissue (GALT) containing the largest pool of immune cells is found (Bourlioux et al., 2003). However, due to the presence of overwhelming potentially immune-stimulatory bacterial and food antigens, this tissue should respond adequately to stimulations. A complex regulation takes place in the gut to allow pathogens recognition while avoiding any unwanted response to the "normal" gut microflora. IECs form the first barrier encountered by luminal antigens and should respond appropriately to have a role in the regulation of immune response (Blikslager et al., 2007). For this purpose, they can secrete chemokines, cytokines and eicosanoids (Mason et al., 2008) to communicate with immune cells and to direct them selectively towards antigens. In particular, IL-8 is an important mediator for these cells, being the major secreted product of infected epithelial cells (Eckmann et al., 1995), e.g. Caco-2 cells (Van De Walle et al., 2010; Sergent et al., 2010). This chemokine produced among others by IECs, has the ability to attract neutrophils to guide them to the site of inflammation and it is thus commonly classified as a pro-inflammatory cytokine (Andoh et al., 2000; Rossi et al., 2013).
As the involvement of oxidative stress in the toxicity of AgNPs has been extensively proven (Völker et al., 2013; Kim and Choi, 2012; Georgantzopoulou et al., 2015; Gaillet and Rouanet, 2015; Akter et al., 2018), we have decided to evaluate whether a transcription factor i.e. Nuclear factor-erythroid 2-related factor (Nrf2) could be involved in this crosstalk as it orchestrates the defence against oxidative stress. Under normal conditions, Nrf2 is sequestrated by the cytosolic protein Keap1, which leads to its proteosomal degradation. Keap1 contains a series of reactive cysteine residues, acting as a sensor through reaction with electrophiles or oxidants. This modification releases Nrf2 that then translocates in the nucleus and regulates the expression of its target genes, containing an antioxidant response element (ARE) in their promoters (Kobayashi and Yamamoto, 2005). They modulate the cellular response to stress such as phase 2 detoxifying enzymes, thiol molecule generating system, reactive oxygen species (ROS) removing enzymes or stress response proteins (Kwak et al., 2004; Kobayashi and Yamamoto, 2005; Kensler et al., 2007). Among these target genes, heme oxygenase-1 (HO-1) is one of the most used to assess the activation of Nrf2 as it has been probably the best characterised ARE (Simmons et al., 2011). This enzyme, also named "heat shock protein 32", catalyzes the heme degradation, releasing free iron, bilirubin and carbon monoxide (Gozzelino et al., 2010). Moderate levels of these three molecules could exert anti-inflammatory and antioxidant properties (Ryter et al., 2006). Indeed, HO-1 seems to have a major role in the protection against acute and chronic inflammation of the gut (Zhu et al., 2011), its induction being associated with a protective response that contributes to the preservation of the gastro-intestinal tract (Zhu et al., 2011). Activation of Nrf2 (Prasad et al., 2013; van der Zande et al., 2016; Mao et al., 2018; Aueviriyavit et al., 2014; Ambrožová et al., 2017; Böhmert et al., 2015) and/or induction of HO-1 (Stępkowski et al., 2014; Bouwmeester et al., 2011; Miura and Shinohara, 2009; Xin et al., 2015; Kang et al., 2012a; Sahu et al., 2015; Ambrožová et al., 2017; Aueviriyavit et al., 2014; Fizesan et al., 2019) have been largely observed as a response to AgNPs.
In this study, we investigated if this Nrf2 cascade could play a role in the secretion of IL-8 mediated by AgNPs as IL-8 displays an ARE in its promoter (Zhang et al., 2005). This study aims at evaluating if AgNPs could modulate the secretion of IL-8 in Caco-2 cells. Moreover, we investigated the involvement of soluble Ag and Nrf2 signalling pathway in this secretion.
Section snippets
Cell culture and exposure
Caco-2 cells from a human colon adenocarcinoma (clone 1 from Dr. M. Rescigno, University of Milano, IT) were cultivated between p + 10 and p + 30 at 37◦C under a water-saturated atmosphere with 10 % (v/v) CO2. Caco-2 cells were grown in tissue culture flasks (Corning incorporated, Corning, NY) in Dulbecco modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Lonza, Basel, CH), supplemented with 10 % (v/v) fetal bovine serum (Biowest, Nuaillé, FR), 1% (v/v) non-essential amino acids 100X (Lonza),
Characterisation of the nanomaterial
The characterisation of dry particles from this batch was performed by Klein et al. (2011) (Klein et al., 2011). We also performed an additional characterisation of AgNPs in HBSS (67.5 μg/mL) by transmission electron microscopy (TEM) (Fig. 2), UV–vis spectrophotometry (Fig. 3A) and dynamic light scattering (DLS). The hydrodynamic diameter measured by DLS was 57.75 nm with a polydispersity index of 0.226. In addition, the absorbance of soluble silver fraction was measured between 350 and 750 nm
Discussion
With their increasing use in food-related consumer products, it is very likely that a certain proportion of these AgNPs are ingested and that they come in contact with IECs from the gut. Although the important role of these cells in immune regulation against luminal potential antigens has been well described, the effect of AgNPs on inflammation in IECs still remains largely unknown. This study analyses the implication of one of the key players of these mechanisms, i.e. IL-8 upon exposure of
Conclusion
In summary, Caco-2 cells produce IL-8 in response to AgNPs exposure. This secretion is polarized towards the luminal compartment and is activated, at least partially, by a Nrf2 dependent pathway suggesting the involvement of oxidative stress in this secretion. This IL-8 secretion seems to be mediated by the soluble fraction composed of Ag + ions present in AgNPs suspensions, suggesting that it is not specific to nanoparticles.
Funding information
The authors declare no conflict of interest. This work has received some financial support from Association Luxembourgeoise des Amis de la Fondation de l’Université Catholique de Louvain (ALAF) and from UCLouvain (FSR Grant).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We would like to thank the SMCS platform from UCLouvain and in particular Vincent Bremhorst and Catherine Rasse for the statistical support.
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