The Comparative Analysis of Cytokine Production by a Human 3D Tissue Model Following Exposure to Traditional Cigarette Smoke, Tobacco-Heated Product and E-Cigarette Aerosol

November 15, 2020

Rob Bedford1, Emma Rothwell1, Sophie Martin1, Cian O’Hanlon1, Andrew McCune2 and Michael Hollings1
1Genetic and Molecular Toxicology and 2Immunology and Immunotoxicology, Covance Laboratories Ltd., Harrogate, UK


– Exposure to 3R4F resulted in increased levels of IL-4, MDC, GM-CSF, IL-12/IL23p40, IL-10 and IFN-γ in the recovery media. Approximately three-fold increases in MDC, GM-CSF, IL-12/IL23p40 and IFNγ were observed whilst two-fold increases were observed for IL-4 and IL-10. In comparison, no marked effect was observed in the module media.

– In contrast to the response observed from 3R4F exposure, fewer changes in cytokine production were observed following THP and E-cigarette exposure. IFNγ demonstrated a two-fold increase in levels measured in the recovery media at doses 1 and 2 for THP. IL-12/IL23p40 also demonstrated a 1.5-fold increase in recovery media following exposure to THP. In addition, IFN-γ and IL-8 were increased following exposure to E-cigarette at dose 2. IL-1β also demonstrated a 1.5-fold increase in the recovery and module media following exposure to E-cigarette.

– A number of cytokines were reduced following exposure to THP and E-cigarette. For example, GM-CSF, MIP1α, VEGF and MCP-1.

– These results demonstrate the difference in cytokine profiles of MucilAir tissues following exposure to different nicotine-containing products.


The battery of regulatory assays currently used to assess the toxicity of aerosol exposure are limited in their ability to identify changes at the cellular and molecular level. This has prompted a shift towards a more holistic systems biology approach when assessing the effects of exposure to potential toxicants.1,2 For example, analysis of the inflammatory mediators produced may provide information on the toxicity-related mechanisms associated with such exposure. 
In this study, we used the V-PLEX® human cytokine kit (MesoScale Diagnostics, LLC) to analyse a panel of 30 disease biomarkers following acute exposure of a human airway 3D tissue model (MucilAirTM, Epithelix Sarl, Switzerland) at the ALI using a Vitrocell® VC10® to cigarette (3R4F) smoke, tobacco-heated product (THP) and Ecigarette aerosol. Following exposure, post-exposure and recovery (24 hour) medias were collected and biomarker levels quantified. Changes were observed for a number of biomarkers in both the post-exposure and post-recovery media including; IL-1β, IL-8, IL-10, IL12p70, IFN-γ and VEGF. For example, IFN-γ demonstrated ~two-fold increase in the post-recovery media following exposure to all test articles, VEGF was decreased following exposure to the THP and e-cigarette products but not 3R4F and IL-1β levels were increased following exposure to 3R4F but decreased following exposure to THP and e-cigarette. Our observations demonstrate the potential of the MSD V-PLEX® human cytokine kit in assessing the effects of aerosol exposure on cytokine production, providing an insight to the different biological pathways affected by different commercially-available nicotine delivery systems.

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