Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response foll
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RESEARCH
Open Access
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 Linda C. Stoehr1,2†, Carola Endes3†, Isabella Radauer-Preiml1, Matthew S. P. Boyles1, Eudald Casals4, Sandor Balog5, Markus Pesch2, Alke Petri-Fink3, Barbara Rothen-Rutishauser3, Martin Himly1, Martin J. D. Clift3 and Albert Duschl1*
Abstract Background: 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. Methods: All 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). Results: 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. Conclusions: 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. Keywords: A549 cells, Interleukin-8, Air-liquid interface, Submerged cultures, Acute pulmonary (pro-)inflammatory effects
* Correspondence: [email protected] † Equal contributors 1 Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria Full list of author information is available at the end of the article © 2015 Stoehr et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Comm
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