Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions

Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm 2 for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures. The online version of this article (doi:10.1186/s12989-015-0104-6) contains supplementary material, which is available to authorized users

Differential immunomodulatory responses to nine polycyclic aromatic hydrocarbons applied by passive dosing

Studying the effects of hydrophobic chemicals using in vitro cell based methods is hindered by the difficulty in bringing and keeping these chemicals in solution. Their effective concentrations are often lower than their nominal concentrations. Passive dosing is one approach that provides defined and stable dissolved concentrations during in vitro testing, and was applied to control and maintain freely dissolved concentrations of polycyclic aromatic hydrocarbons (PAHs) at levels up to their aqueous solubility limit. The immunomodulatory effects of 9 different PAHs at aqueous solubility on human bronchial epithelial cells were determined by analysing the cytoldne promoter expression of 4 different inflammatory cytokines using stably transfected recombinant A549 cell lines. Diverse immunomodulatory responses were found with the highest induction observed for the most hydrophobic PAHs chrysene, benzo(a)antracene and benzo(a)pyrene. Cytokine promoter expression was then studied in dose response experiments with acenaphthene, phenanthrene and benzo(a)anthracene. The strongest induction was observed for benzo(a)anthracene. Cell viability analysis was performed and showed that none of the PAHs induced cytotoxicity at any of the concentrations tested. Overall, this study shows that (1) immunomodulatory effects of PAHs can be studied in vitro at controlled freely dissolved concentrations, (2) the most hydrophobic PAHs were the strongest inducers and (3) induction was often higher at lower exposure levels and decreased then with concentration despite the apparent absence of cytotoxicity. (C) 2014 Elsevier Ltd. All rights reserved

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions

Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm 2 for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures. The online version of this article (doi:10.1186/s12989-015-0104-6) contains supplementary material, which is available to authorized users

Additional file 2 of Nanoparticle-allergen interactions mediate human allergic responses: protein corona characterization and cellular responses

Determination of conjugated allergen after the incubation in human plasma performed by Coomassie-stained SDS-PAGE (A-C) and Western blots (D-F). (TIF 526 kb

Additional file 1 of Nanoparticle-allergen interactions mediate human allergic responses: protein corona characterization and cellular responses

Figure S1. Supplementary material. Control for allergen corona replacement during basophil activation assays. (DOCX 573 kb

Nanotoxicology / Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation

Nanoparticles (NPs) are easily contaminated by bacterial endotoxin (lipopolysaccharide [LPS]). The presence of LPS can be responsible for many immune/inflammatory effects attributed to NPs. In this study, we examined the effects of LPS adsorption on the NP surface on the formation of a biocorona in biological fluids and on the subsequent inflammation-inducing activity of NPs. Different gold (Au) NPs with sizes ranging from 10 to 80 nm and with different surface functionalization (sodium citrate, lipoic acid, and branched polyethyleneimine (BPEI), or polyethylene glycol (PEG)) were exposed to E. coli LPS under different conditions. The binding capacity of LPS to the surface of AuNPs was dose- and time-dependent. LPS attached to sodium citrate and lipoic acid coatings, but did not adhere to BPEI- or PEG-coated NPs. By computational simulation, the binding of LPS to AuNPs seems to follow the Langmuir absorption isotherm. The presence of LPS on AuNP surface interfered and caused a decrease in the formation of the expected biomolecular corona upon incubation in human plasma. LPS-coated AuNPs, but not the LPS-free NPs, induced significant inflammatory responses in vitro. Notably, while free LPS did also induce an anti-inflammatory response, LPS bound to NPs appeared unable to do so. In conclusion, the unintentional adsorption of LPS onto the NP surface can affect the biocorona formation and the inflammatory properties of NPs. Thus, for an accurate interpretation of NP interactions with cells, it is extremely important to be able to distinguish the intrinsic NP biological effects from those caused by biologically active contaminants such as endotoxin

Additional file 1: of Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions

Supplementary Material - Stoehr et al. PFT 2015. (PDF 838 kb

Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation

Nanoparticles (NPs) are easily contaminated by bacterial endotoxin (lipopolysaccharide [LPS]). The presence of LPS can be responsible for many immune/inflammatory effects attributed to NPs. In this study, we examined the effects of LPS adsorption on the NP surface on the formation of a biocorona in biological fluids and on the subsequent inflammation-inducing activity of NPs. Different gold (Au) NPs with sizes ranging from 10 to 80 nm and with different surface functionalization (sodium citrate, lipoic acid, and branched polyethyleneimine (BPEI), or polyethylene glycol (PEG)) were exposed to E. coli LPS under different conditions. The binding capacity of LPS to the surface of AuNPs was dose- and time-dependent. LPS attached to sodium citrate and lipoic acid coatings, but did not adhere to BPEI- or PEG-coated NPs. By computational simulation, the binding of LPS to AuNPs seems to follow the Langmuir absorption isotherm. The presence of LPS on AuNP surface interfered and caused a decrease in the formation of the expected biomolecular corona upon incubation in human plasma. LPS-coated AuNPs, but not the LPS-free NPs, induced significant inflammatory responses in vitro. Notably, while free LPS did also induce an anti-inflammatory response, LPS bound to NPs appeared unable to do so. In conclusion, the unintentional adsorption of LPS onto the NP surface can affect the biocorona formation and the inflammatory properties of NPs. Thus, for an accurate interpretation of NP interactions with cells, it is extremely important to be able to distinguish the intrinsic NP biological effects from those caused by biologically active contaminants such as endotoxin