Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Abstract

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CSexposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

Differential Gene Expression Using RNA Sequencing Profiling in a Reconstituted Airway Epithelium Exposed to Conventional Cigarette Smoke or Electronic Cigarette Aerosols

“…The observed VEGF and IL-8 response at 24 hours postexposure and MCP-1 reduction at 48 hours were in agreement with previous work conducted using the same cell type and a similar short repeated exposure smoking regimen of a 3R4F reference cigarette. 28,34 Furthermore, studies using sample sources such as primary and immortal human bronchial epithelial cells, peripheral blood mononuclear cells, and sputum have shown that cigarette smoke exposure stimulates the expression of MMP-9, IL-8, VEGF, 35 IL-4, 36 and IL-6 37 in the pathogenesis of COPD, which is in agreement with our results.…”

“…These two MMPs and two cytokines are well-documented responders to cigarette smoke in the airways. 28 A significant increase in MMP-1 expression was observed in three donors D1, D3, and D6 in response to whole smoke exposure (Fig. 4).…”

“…Donor 1 was selected for subsequent exposure to 3R4F and Vype ePen based on a consistent MMP-1, MMP-9, and VEGF response that was in good agreement with the literature. 28 Cytotoxicity measured by LDH released on 3R4F exposure remained below 6% for all donors 24 hours postexposure.…”

“…Similar puffing sequences have been reported previously and have been used to represent the intermittent nature of the smoking ritual. 28,33 Furthermore, similar puffing regimens have been applied in vitro to induce markers of respiratory epithelium remodeling observed in the onset of goblet cell hyperplasia and chronic obstructive pulmonary disease (COPD). Those markers include MMPs, VEGF, and IL-8 of which MMP-1, MMP-9, and VEGF gave a relatively consistent response when we exposed MucilAir cells obtained from six donors to 3R4F smoke using the short repeated exposure regimen (Fig.…”

“…4). 28 Since smoking is a cause of chronic inflammation of the respiratory epithelium, we extended the panel of proinflammatory mediators screened to 30 cytokines and 3 MMPs (full list of 33 markers given in Supplementary Table S7) quantified in the culture media of MucilAir cells exposed to 3R4F smoke and Vype ePen aerosol. We reported a significant increase for VEGF, MMP-9, IL-4, IL-6, IL-8, IL-12 p70, IFN gamma, and IL-5 ( p < 0.05, FC >1.5), 24 hours after a short repeated exposure to 3R4F smoke (Fig.…”