The Immunomodulatory Effect of Silver Nanoparticles in a Retinal Inflammatory Environment.

Activation of immune response plays an important role in the development of retinal diseases. One of the main populations of immune cells contributing to the retinal homeostasis are microglia, which represent a population of residential macrophages. However, under pathological conditions, microglia become activated and rather support a harmful inflammatory reaction and retinal angiogenesis. Therefore, targeting these cells could provide protection against retinal neuroinflammation and neovascularization. In the recent study, we analyzed effects of silver nanoparticles (AgNPs) on microglia in vitro and in vivo. We showed that the AgNPs interact in vitro with stimulated mouse CD45/CD11b positive cells (microglia/macrophages), decrease their secretion of nitric oxide and vascular endothelial growth factor, and regulate the expression of genes for Iba-1 and interleukin-1ß (IL-1ß). In our in vivo experimental mouse model, the intravitreal application of a mixture of proinflammatory cytokines tumor necrosis factor-α, IL-1ß and interferon-γ induced local inflammation and increased local expression of genes for inducible nitric oxide synthase, IL-α, IL-1ß and galectin-3 in the retina. This stimulation of local inflammatory reaction was significantly inhibited by intravitreal administration of AgNPs. The application of AgNPs also decreased the presence of CD11b/Galectin-3 positive cells in neuroinflammatory retina, but did not influence viability of cells and expression of gene for rhodopsin in the retinal tissue. These data indicate that AgNPs regulate reactivity of activated microglia in the diseased retina and thus could provide a beneficial effect for the treatment of several retinal diseases.

The Impact of Metal Nanoparticles on the Immunoregulatory and Therapeutic Properties of Mesenchymal Stem Cells.

Negative impacts of nanomaterials on stem cells and cells of the immune system are one of the main causes of an impaired or slowed tissue healing. Therefore, we tested effects of four selected types of metal nanoparticles (NPs): zinc oxide (ZnO), copper oxide (CuO), silver (Ag), and titanium dioxide (TiO2) on the metabolic activity and secretory potential of mouse mesenchymal stem cells (MSCs), and on the ability of MSCs to stimulate production of cytokines and growth factors by macrophages. Individual types of nanoparticles differed in the ability to inhibit metabolic activity, and significantly decreased the production of cytokines and growth factors (interleukin-6, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor-1) by MSCs, with the strongest inhibitory effect of CuO NPs and the least effect of TiO2 NPs. The recent studies indicate that immunomodulatory and therapeutic effects of transplanted MSCs are mediated by macrophages engulfing apoptotic MSCs. We co-cultivated macrophages with heat-inactivated MSCs which were untreated or were preincubated with the highest nontoxic concentrations of metal NPs, and the secretory activity of macrophages was determined. Macrophages cultivated in the presence of both untreated MSCs or MSCs preincubated with NPs produced significantly enhanced and comparable levels of various cytokines and growth factors. These results suggest that metal nanoparticles inhibit therapeutic properties of MSCs by a direct negative effect on their secretory activity, but MSCs cultivated in the presence of metal NPs have preserved the ability to stimulate cytokine and growth factor production by macrophages.

Effects of antimicrobial metal nanoparticles on characteristics and function properties of mouse mesenchymal stem cells.

Nanoparticles (NPs) have a wide use in various field of industry and in medicine, where they represent a promise for their antimicrobial effects. Simultaneous application of NPs and therapeutic stem cells can speed up tissue regeneration and improve healing process but there is a danger of negative impacts of NPs on stem cells. Therefore, we tested effects of four types of metal antimicrobial NPs on characteristics and function properties of mouse mesenchymal stem cells (MSCs) in vitro. All types of tested NPs, i.e. zinc oxide, silver, copper oxide and titanium dioxide, exerted negative effects on the expression of phenotypic markers, metabolic activity, differentiation potential, expression of genes for immunoregulatory molecules and on production of cytokines and growth factors by MSCs. However, there were apparent differences in the impact of individual types of NPs on tested characteristics and function properties of MSCs. The results showed that individual types of NPs influence the activity of MSCs, and thus the use of metal NPs during tissue regeneration and in combination with stem cell therapy should be well considered.

The impact of extractable organic matter from gasoline and alternative fuel emissions on bronchial cell models (BEAS-2B, MucilAir™).

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAir™). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

In vitro exposure to complete engine emissions - a mini-review.

Outdoor air pollution is classified as carcinogenic to humans and exposure to it contributes to increased incidence of various diseases, including cardiovascular, neurological or pulmonary disorders. Vehicle engine emissions represent a significant part of outdoor air pollutants, particularly in large cities with high population density. Considering the potentially negative health impacts of engine emissions exposure, the application of reliable test systems allowing assessment of the biological effects of these pollutants is crucial. The exposure systems should use relevant, preferably multicellular, cell models that are treated with the complete engine exhaust (i.e. a realistic mixture of particles, chemical compounds bound to them and gaseous phase) at the air-liquid interface. The controlled delivery and characterization of chemical and/or particle composition of the exhaust should be possible. In this mini-review we report on such exposure systems that have been developed to date. We focus on a brief description and technical characterization of the systems, and discuss the biological parameters detected following exposure to a gasoline/diesel exhaust. Finally, we summarize and compare findings from the individual systems, including their advantages/limitations.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

Genotoxicant exposure, activation of the aryl hydrocarbon receptor, and lipid peroxidation in cultured human alveolar type II A549 cells.

The aryl hydrocarbon receptor (AhR) transcription factor is activated by polycyclic aromatic hydrocarbons (PAH) and other ligands. Activated AhR binds to dioxin responsive elements (DRE) and initiates transcription of target genes, including the gene encoding prostaglandin endoperoxide synthase 2 (PTGS-2), which is also activated by the transcription factor NF-ĸB. PTGS-2 catalyzes the conversion of arachidonic acid (AA) into prostaglandins, thromboxanes or isoprostanes. 15-F2t-Isoprostane (IsoP), regarded as a universal marker of lipid peroxidation, is also induced by PAH exposure. We investigated the processes associated with lipid peroxidation in human alveolar basal epithelial cells (A549) exposed for 4 h or 24 h to model PAH (benzo[a]pyrene, BaP; 3-nitrobenzanthrone, 3-NBA) and organic extracts from ambient air particulate matter (EOM), collected in two seasons in a polluted locality. Both EOM induced the expression of CYP1A1 and CYP1B1; 24 h treatment significantly reduced PTGS-2 expression. IsoP levels decreased after both exposure periods, while the concentration of AA was not affected. The effects induced by BaP were similar to EOM except for increased IsoP levels after 4 h exposure and elevated AA concentration after 24 h treatment. In contrast, 3-NBA treatment did not induce CYP expression, had a weak effect on PTGS-2 expression, and, similar to BaP, induced IsoP levels after 4 h exposure and AA levels after 24 h treatment. All tested compounds induced the activity of NF-ĸB after the longer exposure period. In summary, our data suggest that EOM, and partly BaP, reduce lipid peroxidation by a mechanism that involves AhR-dependent inhibition of PTGS-2 expression. The effect of 3-NBA on IsoP levels is probably mediated by a different mechanism independent of AhR activation.

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAirTM) and in Human Bronchial Epithelial Cells (BEAS-2B).

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAirTM) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected CYP1A1 and HSPA5 expression in MucilAirTM. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.

DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells.

Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 µg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 µM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 µM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 µM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 µM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system.