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

Abstract

BackgroundStably 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.MethodsAll cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm2 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).ResultsIn 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0104-6) contains supplementary material, which is available to authorized users.

Dry Generation of CeO2 Nanoparticles and Deposition onto a Co-Culture of A549 and THP-1 Cells in Air-Liquid Interface—Dosimetry Considerations and Comparison to Submerged Exposure

“…Other studies have also compared ALI and submerged conditions with diverging results [17,18,23,25]. Our study can be directly compared to the study performed by Loret et al (2016), which used a very similar approach for toxicity testing the same CeO 2 NPs (NM-212) [17].…”

“…These systems have previously been used to generate dry aerosols and expose cultured cells to palladium (Pd) NPs [20], and diesel exhaust particles [21], as well as carbonaceous model (Printex 90) NPs [22]. A general difficulty in comparing different exposure set-ups is the dose comparison [23]. To allow for direct comparisons between these two exposure methods, the cells were cultured at similar conditions (on inserts), and cell doses were analyzed quantitatively (using ICP-MS) and expressed as µg/cm 2 for both ALI and submerged exposures.…”

The curious case of how mimicking physiological complexity in in vitro models of the human respiratory system influences the inflammatory responses. A preliminary study focused on gold nanoparticles

“…Nevertheless, the majority of the risk assessment studies on inhaled NPs is performed on submerged monocultures grown on glass or plastic substrates. Several publications have demonstrated that co‐cultures of multiple cell types have a strong influence on the observed cellular responses to inhaled NPs (Blank et al, ; Clift et al, ; Lehmann et al, ; Muller et al, ; Rothen‐Rutishauser, Blank, et al, ; Rothen‐Rutishauser, Mueller, et al, ; Rothen‐Rutishauser et al, ; Stoehr et al, ). Thus, in our case study, four different in vitro models of the lung epithelium were tested in order to quantify whether the in vitro complexity and culturing substrates can affect the detectable pulmonary inflammatory response to AuNPs.…”

A guide to nanosafety testing: Considerations on cytotoxicity testing in different cell models

“…For instance, surfactant can be used in the case of lung models (McKenzie et al 2015;Raesch et al 2015) and for oral uptake the different consecutive pH values encountered along the stages of the gastro-intestinal tract can be simulated (Peters et al 2012). Furthermore, skin and lung models may be exposed at the airliquid interface (Stoehr et al 2015) while ingestion models could include a more realistic digestion fluid on one side. Finally, cell models are being refined, and new developments such as microfluidic systems and organoids promise to allow more realistic models and to eliminate the species differences in toxicological data by using human cells (Esch et al 2014;Tralau et al 2015).…”