Multi-endpoint analysis of human 3D airway epithelium following repeated exposure to whole electronic vapor product aerosol or cigarette smoke

Abstract

Smoking is a cause of serious diseases in smokers including chronic respiratory diseases. This study aimed to evaluate the tobacco harm reduction (THR) potential of an electronic vapor product (EVP, myblu™) compared to a Kentucky Reference Cigarette (3R4F), and assessed endpoints related to chronic respiratory diseases. Endpoints included: cytotoxicity, barrier integrity (TEER), cilia function, immunohistochemistry, and pro-inflammatory markers. In order to more closely represent the user exposure scenario, we have employed the in vitro 3D organotypic model of human airway epithelium (MucilAir™, Epithelix) for respiratory assessment. The model was repeatedly exposed to either whole aerosol of the EVP, or whole 3R4F smoke, at the air liquid interface (ALI), for 4 weeks to either 30, 60 or 90 puffs on 3-exposure-per-week basis. 3R4F smoke generation used the ISO 20778:2018 regime and EVP aerosol used the ISO 20768:2018 vaping regime. Exposure to undiluted whole EVP aerosol did not trigger any significant changes in the level of pro-inflammatory mediators, cilia beating function, barrier integrity and cytotoxicity when compared with air controls. In contrast, exposure to diluted (1:17) whole cigarette smoke caused significant changes to all the endpoints mentioned above. To our knowledge, this is the first study evaluating the effects of repeated whole cigarette smoke and whole EVP aerosol exposure to a 3D lung model at the ALI. Our results add to the growing body of scientific literature supporting the THR potential of EVPs relative to combustible cigarettes and the applicability of the 3D lung models in human-relevant product risk assessments.

Keywords: 2D, Two Dimensional; 3D, Three Dimensional; 3R4F, Scientific Reference Tobacco Cigarette (University of Kentucky); ALI, Air-Liquid Interface; ANOVA, Analysis of Variance; AOP, Adverse Outcome Pathway; CAA, Cilia Active Area; CBF, Cilia Beat Frequency; COPD, Chronic Obstructive Pulmonary Disease; CYP450, Cytochrome P450; Cigarette; Cilia; DPBS, Dulbecco's phosphate-buffered saline containing Ca2+ and Mg2+; EGFR, Epidermal Growth Factor Receptor; EVP, Electronic Vapor Product; Electronic vapor product; FOX-J1, Forkhead Box J1 protein; H&E, Hematoxylin and Eosin; IIVS, Institute for In Vitro Sciences; IL-13, Interleukin 13; IL-1β, Interleukin 1 Beta; IL-6, Interleukin-6; IL-8, Interleukin-8; ISO, International Organization for Standardization; Immunohistochemistry; KERs, Key Event Relationships; KEs, Key Events; LDH, Lactate Dehydrogenase; MIE, Molecular Initiating Event; MMP-1, Matrix Metalloproteinase-1; MMP-3, Matrix Metalloproteinase-3; MMP-9, Matrix Metalloproteinase-9; MUC5AC, Mucin 5AC Protein; MWP, Multi-Well Plate; NKT, Natural Killer T Cells; Organotypic tissue model; PBS, Phosphate Buffered Saline; PMN, polymorphonuclear; Pro-inflammatory markers; SAEIVS, Smoke Aerosol Exposure In Vitro System; TEER, Transepithelial Electrical Resistance; THR, Tobacco Harm Reduction; TNF-α, Tumor Necrosis Factor Alpha; TPM, Total Particulate Matter.

Graphical abstract

Fig. 1

Schematic diagram of the experimental design, depicting which experimental steps was carried on each specific test day (T) from day −5 (before the first time of exposure) to 28. * T19, Immunohistochemistry was carried out only for tissues exposed to IL- 13.

Fig. 2

Mean concentration of whole smoke and myblu aerosol nicotine delivery to the exposure chambers by quantification of nicotine in blank well MWP containing PBS. The individual values are a mean of 8 repeats per puff dose. Mean values for concentration are plotted above the corresponding bar. Key to significance. **** p ≤ 0.0001. Error bars are the standard deviation.

Fig. 3

The 3R4F smoke and the myblu aerosol cytotoxicity assessment quantifying LDH levels at different time points in the culture medium over the 28 days. Key to significance. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.0001. N = 3 per time point. Error bars are the standard error of the mean.

Fig. 4

Heat-maps of the differences in expression of inflammatory markers, calculated using the difference between log intensity of the test product and the control (log fold change). A total of six heat-maps are plotted, corresponding to three treatment conditions (30, 60 & 90 puffs) with diluted 3R4F reference cigarette smoke or undiluted myblu aerosol. Analysis of pro-inflammatory markers from T23-28 were excluded for 90 puffs of 3R4F smoke, due to significantly high levels of cytotoxicity. Each map represents the log-ratio of the seven inflammatory mediators (y-axis) measured over a period of 28 days (x-axis). The color represents the change in expression of the inflammatory mediator compared to control. Expression level is plotted according to a continuous color scale, indicated in the ‘Color key’, provided at the right of the figure. Red-scale color represents an inflammatory mediator over-expression, whilst blue-scale color represents an inflammatory mediator under-expression relative to the air control. White color corresponds to a log fold change equal to zero, i.e., no difference in expression observed between the control and the product assessed. The significance threshold in the statistical test was adjusted to 5% (p-value < 0.05), and significant responses are indicated by ‘+’. Note that the MMP-3 at T0 is deemed significantly different from air control, this is due to the very low in-treatment group variability, where even a small change was deemed significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

TNF-α, MMP-9, MMP-3, and MMP-1 levels in culture media over 28 days during which repeated exposures of whole aerosol/smoke (1:17 dilution) /humidified air were applied. Analysis of pro-inflammatory markers from T23-28 were excluded for 90 puffs of 3R4F smoke, due to significantly high levels of cytotoxicity. Levels are expressed as fold change compared to air control response. Note the y-axis scale differences between 3R4F and myblu graphs. Key to significance. +, indicates a significant difference where the adjusted p < 0.05. Error bars are the standard error of the mean.

Fig. 6

IL-1β, IL-6 and IL-8 levels in culture media over 28 days during which repeated exposures of whole aerosol/smoke (1:17 dilution)/humidified air were applied. Analysis of pro-inflammatory markers from T23-28 were excluded for 90 puffs of 3R4F smoke, due to significantly high levels of cytotoxicity. Levels are expressed as fold change compared to air control response. Note the y-axis scale differences between 3R4F and myblu graphs. Key to significance. +, indicates a significant difference where the adjusted p < 0.05. Error bars are the standard error of the mean.

Fig. 7

Representative immunohistochemistry images (alcian blue + H&E, MUC-5AC and FOX-J1) of 3D lung models after 28 days of repeated exposure to 30, 60 or 90 puffs of myblu aerosol (undiluted), 3R4F cigarette smoke (dilution 1:17 smoke: air) or negative control (90 puffs of humidified filtered air). Tissues exposed to IL-13 was evaluated at T19. x̄ in table (Quantified staining average). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Fold change of CBF and CAA over time (T0 - T28) following repeated exposures to 30, 60 or 90 puffs of 3R4F smoke (A &B) and myblu aerosol (C & D) compared to matching air controls. Key to significance. Key to significance. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.0001. Error bars are the standard error of the mean. Analysis of CBF and CAA were excluded for 90 puffs of 3R4F smoke from T23-28 due to significantly high levels of cytotoxicity (see Fig. 3).

Fig. 9

Fold change of 3D lung TEER value over time (T0 - T28) following repeated exposures to 30, 60 or 90 puffs of 3R4F smoke (A) and myblu aerosol (B) compared to matching air controls. Key to significance. ** p ≤ 0.01; **** p ≤ 0.0001. Error bars are the standard error of the mean. To note, significantly high levels of cytotoxicity for the 90 puffs of 3R4F smoke was observed from T23-28 (see Fig. 3).

Fig. 10

The progression of adverse events following exposure at the ALI based on Peitsch et al., 2018.