Using Lifeline® Lung-Airway Cells to Study Viral Infection

The Lung-Airway Epithelium

When air enters the respiratory system, it travels through the trachea and into one of the two bronchi, which lead to either lung as they branch into bronchioles. Finally, in the lung, the bronchioles terminate in alveoli, small sacs in which gas exchange occurs. The entire airway is lined by epithelial cells that serve as a barrier to the external environment, which may contain various respiratory viruses, such as rhinoviruses or coronaviruses.

The airway epithelium is the site of respiratory viral infection, which can be readily studied in vitro. In particular, Lifeline® bronchial/tracheal epithelial cells (described in a study below) can be cultured in an air-liquid interface in Air-Liquid Interface Epithelial Differentiation Medium, which allows differentiation into an airway-like epithelial monolayer that can be used to study cell differentiation and mechanisms of viral infection.

Using Lifeline® Lung-Airway Cells to Study Viral Infection: Recent Studies

Human coronaviruses (CoVs) are a major cause of respiratory illness worldwide. CoV infection occurs upon interaction of the viral spike (S) protein with a receptor on the surface of airway epithelial cells. Understanding how the S protein binds its receptor is critical for determining how to target and disrupt that interaction to treat CoV infection. To that end, Ou et al. (2017) solved a crystal structure of the human HKU1 beta CoV S protein C-terminal domain, which binds its currently unknown receptor. The authors identified three domains within the crystal structure: the core, the insertion loop, and the subdomain (SD)-1. Unlike the core and SD-1 domains, which are structurally similar to other beta CoVs, the insertion loop was unique.

To begin to understand how the HKU1 S protein might interact with its receptor, the authors integrated their structure with a previously described electron microscopic structure, which includes more of the protein. Interestingly, they found that the structurally analogous receptor binding motifs in HKU1 (compared to the S protein structures of other family members) were buried, suggesting that conformational changes might be required to unmask them for receptor binding.

Additionally, using various neutralizing antibodies, the authors mapped the neutralizing epitopes to the tip of the S protein. Finally, using Lifeline® human bronchial/tracheal epithelial cells grown as a monolayer in an air-liquid interface, the group evaluated the infection capacity of different S protein point mutants. Importantly, this analysis revealed that residues W515 and R517 are required for receptor binding. Together, the results of this study provide information on how the HKU1 S protein potentially interacts with its receptor.

The ability to exogenously express genes and proteins in experimental cell systems often depends on viral infection. Replication-defective adenoviruses are often used to deliver transgenes to cells, but are limited in their ability to amplify transgene expression. However, replication-competent adenoviruses, while able to significantly amplify transgene expression, are also highly infectious. To overcome this problem, Crosby et al. developed single cycle adenoviruses, which are able to readily replicate their DNA (and the associated transgene), but are unable to propagate infectious viral progeny. The authors began characterizing the three types of adenoviruses—replication-deficient (RD), replication-competent (RC), and single cycle (SC)—using Lifeline® human small airway epithelial cells. They found that adenovirus-mediated GFP transgene expression occurred faster with RC and SC than RD, but was associated with significantly more cytotoxicity.

Although they were similarly effective in vitro, the authors set out to characterize the viruses in a more clinically relevant setting. Therefore, using a reporter system in monocytes, the authors evaluated the capacity of the different viruses to induce interferon-stimulated genes, which is an important anti-viral immune response. They found that RC adenovirus significantly induced the interferon response compared to SC and RD.

Finally, the authors compared these adenoviruses in vivo and found that the efficacy of viral infection was dependent on the route of infection and the immune status of the mouse model. For example, they found that when delivered intravenously into immunocompetent mice, the RC adenovirus was better than SC, and both were better than RD. In contrast, when delivered intranasally, SC was better than RC, and both were better than the RD virus, although the responses of all three viruses were significantly weaker than the intravenous route. When delivered by both routes to immunodeficient mice, the authors found that SC induced transgene expression most effectively, compared to RC or RD viruses. Together, this study presents a direct comparison of three adenoviruses and demonstrates the utility of the newly developed SC adenovirus, which is a promising tool for the development of new vaccines and viral-based therapies.

The Lifeline® catalog of lung/airway cells and their recommended associated growth media includes:

• Small Airway Epithelial Cells (with BronchiaLife™ Medium)
• Lung Microvascular Endothelial Cells (with VascuLife® Medium)
• Bronchial/Tracheal Epithelial Cells (with BronchiaLife™ Medium and Air-Liquid Interface Epithelial Differentiation Medium)
• Lung Fibroblasts (with FibroLife® Medium)
• Bronchial/Tracheal Smooth Muscle Cells (with VascuLife® SMC Medium)
• Lung Smooth Muscle Cells (with VascuLife® Medium)
• Lobar Bronchial Epithelial Cells (with BronchiaLife™ Medium)

Keep in touch with us! We are on the lookout for new feature studies for our blog. Tell us how you are using Lifeline® cells and your work could be highlighted here!