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 Ī¼g/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-Ī± treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-Ī±, 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.
Highlights
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
Fendyur and colleagues assessed the ability for Ag-NPs to induce reactive oxygen species (ROS)-associated DNA damage in NIH-3 T3 cells [15], whilst Karlsson et al [16] investigated the impact of copper (CuO), zinc (ZnO) and nickel oxide (NiO) NP exposure on mouse embryonic stem cells using green fluorescent protein (GFP) to quantify DNA damage and oxidative stress associated with metal oxideinduced cytotoxicity
It cannot be ruled out that some NPs remained in the supernatant after centrifugation, possibly contributing a particle effect to the observed responses, these findings suggest that the zinc oxide (ZnO)-NPs effects on IL-8 expression were primarily caused by ions, which is, as the agglomeration mentioned above, an issue to be considered for submerged exposure
Summary
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. Several studies have reported the use of luciferase reporter cell lines to assess cellular immune modulation in response to gold (Au), carbon, silver (Ag), silica (SiO2) and metal(āoxide) NPs [9,10,11,12,13] and some have validated the observed promoter activity with conventional methods (e.g. qRT-PCR and ELISA), showing good correlation between reporter assays and secreted cytokine analysis [7, 10, 11]. As demonstrated by Kohl et al [17], it was possible to culture pIL8-GFP-A549 cells within a novel micro-culture chamber and subsequently deduce the pro-inflammatory responses to Au, Ag, and magnetite NPs with a microscopy-based approach at the single-cell level
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