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

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/cm(2) 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.

Fig. 1

Evaluating cytotoxicity of ZnO nanoparticles under submerged and ALI conditions. A. Cytotoxicity as determined by LDH release assay. Data are presented as mean x-fold increase over untreated control (submerged) or NaCl-nebulized control (ALI) (dashed line). Error bars indicate the SEM of at least three independent experiments. A one-way analysis of variance (ANOVA) with a subsequent Tukey’s Multiple Comparison test was performed. Values were considered significantly different compared to the unexposed (submerged) or NaCl-nebulized (ALI) control or as indicated with p < 0.05 (*), p < 0.001 (**) and p < 0.0001 (***)

Fig. 2

Pro-inflammatory response upon exposure to ZnO-NPs monitored by pIL8 A549 reporter cell lines under submerged and ALI conditions. Data are presented as mean x-fold increase over untreated control (submerged) or NaCl-nebulized control (ALI) (dashed line). Error bars indicate the SEM of at least three independent experiments. A one-way analysis of variance (ANOVA) with a subsequent Tukey’s Multiple Comparison test was performed. Values were considered significantly different compared to the unexposed (submerged) or NaCl-nebulized (ALI) control or as indicated with p < 0.05 (*), p < 0.001 (**) and p < 0.0001 (***); Ns = not significant

Fig. 3

IL8 gene expression upon exposure to ZnO-NPs under submerged and ALI conditions monitored by qRT-PCR. Data are presented as mean x-fold increase over untreated control (submerged) or NaCl-nebulized control (ALI) (dashed line). Error bars indicate the SEM of at least three independent experiments. A one-way analysis of variance (ANOVA) with a subsequent Tukey’s Multiple Comparison test was performed. Values were considered significantly different compared to the unexposed (submerged) or NaCl-nebulized (ALI) control or as indicated with p < 0.05 (*), p < 0.001 (**) and p < 0.0001 (***)

Fig. 4

Secretion of IL-8 upon exposure to ZnO-NPs under submerged and ALI conditions monitored by ELISA. Data are presented as mean x-fold increase over untreated control (submerged) or NaCl-nebulized control (ALI) (dashed line). Error bars indicate the SEM of at least three independent experiments. A one-way analysis of variance (ANOVA) with a subsequent Tukey’s Multiple Comparison test was performed. Values were considered significantly different compared to the unexposed (submerged) or NaCl-nebulized (ALI) control or as indicated with p < 0.05 (*), p < 0.001 (**) and p < 0.0001 (***)