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

Stoehr, Linda C.; Endes, Carola; Radauer-Preiml, Isabella; Boyles, Matthew S. P.; Casals, Eudald; Balog, Sandor; Pesch, Markus; Petri-Fink, Alke; Rothen-Rutishauser, Barbara; Himly, Martin; Clift, Martin J. D.; Duschl, Albert

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.

• Journal Article Type: Article
• Acceptance Date: Sep 18, 2015
• Online Publication Date: Sep 29, 2015
• Publication Date: 2015
• Deposit Date: Oct 13, 2023
• Publicly Available Date: Oct 16, 2023
• Journal: Particle and Fibre Toxicology
• Print ISSN: 1743-8977
• Publisher: BMC
• Peer Reviewed: Peer Reviewed
• Volume: 12
• Article Number: 29
• DOI: https://doi.org/10.1186/s12989-015-0104-6
• Keywords: A549 cells, Interleukin-8, Air-liquid interface, Submerged cultures, Acute pulmonary (pro-)inflammatory effects