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*
1Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
2Grimm Aerosol Technik GmbH & Co. KG, Ainring, Germany.
3BioNanomaterials, Adolphe Merkle Institute, Université de Fribourg, Fribourg, Switzerland.
4Institut Català de Nanotecnologia (ICN), Bellaterra, Spain.
5Soft Matter Scattering, Adolphe Merkle Institute, Université de Fribourg, Fribourg, Switzerland.
* Correspondence: firstname.lastname@example.org
The use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in the air-liquid interface. The results are shown in the IL-8 assay, qRT-PCR ans ELISA.
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.