Item Details

title

Development of a Biomimetic 3D Airway Organ Tissue Equivalent Model to Understand Aerosolized Toxicant Exposures and Their Influence on Viral Infection

author

Leach, Timothy

abstract

Pulmonary diseases and respiratory infections represent a significant global health challenge contributing to an estimated 4 million premature deaths each year. Tobacco use is a major risk factor for a range of pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Beyond the development of chronic diseases, tobacco use has been shown to increase the susceptibility of the lungs to acute respiratory infection. As the tobacco industry continues to evolve with the introduction of novel tobacco products (NTPs), such as electronic cigarettes or e-cigarettes (ECs), a heightened level of concern has emerged about their impact on pulmonary health. Understanding the mechanisms behind the relationship between tobacco use, pulmonary disease, and infection requires complex models with cell-cell and cell-microenvironment interactions. Current two-dimensional (2D) cell culture models are unable to fully replicate the in vivo microenvironment resulting in concerns over translational potential. In recent years, there have been significant advancements in three-dimensional (3D) cell culture techniques, resulting in more complex in vitro models that better replicate the microenvironment of human tissues providing platforms to study these intricate cell interactions. However, many of the current 3D airway models are limited by one or more of the following: the lack of an air-liquid interface (ALI) that is characteristic of the airway epithelium, the lack of multiple cell types resident to airway tissue, and a 3D microenvironment that is inconsistent in extracellular matrix (ECM) composition and biomechanical properties compared to native in vivo airway tissue. The work presented in this dissertation focuses on the development of a human airway organ tissue equivalent (OTE) model comprising multi-cell types and a 3D microenvironment more characteristic of in vivo airway tissue that can be maintained at ALI for toxicological and infection studies, specifically regarding tobacco products. This thesis is comprised of 3 aims:1) Development of a multi-cell type airway OET in vitro ALI model. We developed and characterized an airway 3D cell culture model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. Our hypothesis posited that the inclusion of native lung fibroblasts, lung-specific ECM, and the fine-tuning of hydrogel biomechanical properties within a physiological range would enhance the maturation of human bronchial epithelial cells. The OTEs that incorporated native lung fibroblasts and lung extracellular matrix provided a microenvironment that promoted the maturation of bronchial epithelium and maintained a barrier function, while hydrogel stiffness could be adjusted while maintaining epithelial differentiation. The results highlight the versatility of the OTE model by providing the ability to alter physiologically relevant microenvironment parameters while maintaining ALI. 2) Analysis of the acute exposure and toxicity of combustible and novel tobacco products (NTPs) using an airway OTE model. We hypothesized that NTPs, such as ECs and heated tobacco products (HTPs), despite having reduced cytotoxicity compared to cigarettes, would still exert a substantial impact on the OTE’s inflammatory profile. We compared different NTPs to a commercially available cigarette product normalized to nicotine delivered per product to the OTE tissue. With our airway OTE model, our data illustrated a reduction in toxicity from the NTPs in comparison to the cigarette product although the HTP produced a notable inflammatory response and negative effects on epithelial barrier and cilia function. The EC products did not induce significant changes in inflammation or epithelial function, but did alter some gene expression, emphasizing effects beyond cytotoxicity. This study highlights the necessity of investigating the effects of NTPs beyond cytotoxicity and inflammation in comparison to cigarettes. 3) Analysis of the susceptibility of airway OTEs to viral infection post-exposure to aerosolized tobacco products. We hypothesized that exposure to tobacco products, specifically a cigarette and EC, would inhibit the OTE’s antiviral defense mechanisms, leading to heightened viral replication and load. Inhalation of tobacco products, particularly cigarettes, has been directly linked to increased susceptibility to infection. We examined the susceptibility of the airway OTE to influenza A virus (IAV) following acute exposure to aerosolized tobacco products. We explored the timeline of infection with regard to viral load and antiviral response of the airway OTE tissue from 6 hours to 48 hours post-infection (hpi). Our results indicate distinct alterations in the airway OTE’s response to IAV infection post-exposure to the tobacco products. Cigarette exposure led to a reduction in antiviral genes, likely leading to a delayed antiviral response and heightened viral load after 48 hpi compared to control samples. In contrast, e-cigarette exposure downregulated genes associated with viral endocytosis, possibly resulting in decreased viral load and a diminished antiviral response. Further investigations into the magnitude of these effects with viral infection should be explored.

subject

3D cell culture

air-liquid interface

airway

novel tobacco products

organoid

contributor

Atala, Anthony (advisor)

Files, Daniel Clark (committee member)

Hall, Adam (committee member)

Soker, Shay (committee member)

date

2024-02-13T09:36:07Z (accessioned)

2024 (issued)

degree

Biomedical Engineering (discipline)

embargo

2029-01-12 (terms)

2029-01-12 (liftdate)

identifier

http://hdl.handle.net/10339/102911 (uri)

language

en (iso)

publisher

Wake Forest University

type

Dissertation