Mechanisms of virus infection of human airway epithelium cultured at an air-liquid interface (HAE-ALI)

ABSTRACT Polarized human airway epithelial (HAE) cultures are instrumental for studying respiratory tract diseases and gene delivery of airways. Primary epithelial cells isolated from tracheobronchial airways, grown in transwell at an air-liquid interface (ALI), undergo media-induced mucociliary differentiation. This process reproduces a bronchi tissue-like structure, resembling the morphology and physiology of human airways. HAE-ALI is composed of ciliated, basal, club, and goblet cells, exhibiting features of epithelial barrier, such as intact tight junctions and confluent cilia. Recombinant adeno-associated virus (rAAV) vector is a powerful tool used to treat human genetic diseases; however, a robust research model is critical for developing gene therapy approaches. There's a pressing need for cell culture and animal models that can be used to assess rAAV transduction and predict the efficacy of the vectors in human clinical trials. HAE-ALI cultures derived from immortalized human airway epithelial cells provide an invaluable platform for evaluating the role of host genes in rAAV transduction efficiency in airways, by using CRIPSR/Cas9 gene knockout. In addition, it's noteworthy that rAAV transduction efficiency varies between the cells of human organs and other animal models, emphasizing the importance of cell line-derived HAE-ALI cultures in evaluating novel airway tropic AAV vectors. we identified several critical host factors for AAV2.5T transduction in HAE-ALI and revealed the mechanism of these host factors during rAAV2.5T transduction. HAE-ALI is also a useful tool for studying infection of respiratory viruses, such as SARS-CoV-2 and influenza virus. Understanding SARS-CoV-2 infection of HAE-ALI and the caused epithelial damage has significance and implications in developing effective antivirals. we found that SARS-CoV-2 infection is a multi-step process requiring its receptor ACE2, which is predominantly expressed in airway epithelial cells, and therefore HAE-ALI is an ideal model for in vitro studies of SARS-CoV-2 to understand how the virus infects and damages airway structure and barrier function. We generated HAE-ALI cultures from primary bronchial epithelial cells and used them to model SARS-CoV-2 infection over a long time (up to 51 days), focusing on viral replication kinetics, infection dose-dependency, epithelial damage, and permissive cell subpopulations. We discovered that SARS-CoV-2 prefers to infect HAE-ALI cultures from the apical surface. Importantly, the infection persisted, with recurrent virus release peaks from the infected ciliated and goblet cells, while airway basal and club cells remained nonpermissive. The study revealed that SARS-CoV-2 infection induces recurrent airway epithelial damage, including tight-junction disruptions, reduced cilia, and epithelial cell hypertrophy. Using the HAE-ALI model, we discovered that necroptosis is the key cell death pathway in the damage of the epithelial barrier, and silencing of genes involved in necroptosis ameliorated airway epithelial damage during SARS-Co-2 infection. Apparently, the HAE-ALI model offers valuable insights into the mechanism of rAAV transduction of cells from the human airway and how SARS-CoV-2 replication causes cell death of airway epithelial cells. This knowledge will be crucial for airway gene therapy and effectively combating COVID-19.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2024

Author dc:creator

• Hao, Siyuan

Advisor dc:contributor.advisor

• Qiu, Jianming J.Q.

• Medicine
• Virology
• Microbiology
• AAV
• Human Airway Epithelia
• SARS-CoV-2

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:19353

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/38358