The Airway Epithelium and Air-Liquid Interface Culture

A single later of epithelial cells lines many of our internal organs, including the airway. The airway epithelium is composed of multiple differentiated cell types that form a barrier against pathogens and irritants that are inhaled from the environment. Ciliated cells are so named for the hair-like projections on their surface that serve to push mucus secretions out of the airway. Goblet cells secrete mucus, which serves to trap debris and pathogens, which are in turn driven out by the actions of the ciliated cells. Finally, the basal cells are thought to be the progenitor cells of the airway epithelium, providing a pool of multipotent cells that replace the population of differentiated cells.

It is becoming well appreciated that culturing cells on plastic is not relevant to their endogenous environment. In vivo, airway epithelial cells contact a basement membrane and interact with immune cells and smooth muscle cells to perform their whole tissue function. In vitro, it is difficult to mimic that complex environment, but advances in cell culture techniques have made progress.

Air-liquid interface culture is a common way to model the airway in vitro. This culture system involved culturing airway epithelial cells on porous inserts such that the basal membrane of the cells is in contact with medium and the apical surface of the cells is exposed to air. Using this more physiologically relevant system, the authors of the study described below compare the infectivity of clinically isolated viral strains and their long-term cultured counterparts.

The Lifeline® catalog of airway and lung cells, and media includes:

Lifeline® Airway Epithelial Cells in Current Viral Research

Human coronaviruses (HCoVs) are the main cause of the common cold. They infect airway epithelial cells, which make more viruses and perpetuate the infection. HCoV infection is regulated by activation of the viral spike (S) protein, which is a glycoprotein on the outside of a viral particle that recognizes receptors on host cells. S proteins are activated via cellular proteases, either on the cell surface or within the endosome.

In a previous study, Shirato and colleagues reported that a clinical isolate of HCoV-229E used the cell surface protease TMPRSS2 to activate its S protein, while lab isolates of HCoV-299E preferred the late endosome pathway using cathepsin L. This study suggested that the long-term culture of HCoV-229E lab isolates changed the behavior of the virus over time.

Since their previous study raised concerns about the clinical relevance of laboratory strains, the same group set out to determine whether this mechanistic alteration of S protein activation was also true in other HCoV strains. First, the researchers isolated HCoV samples from patient nasal swabs. They were able to isolate four HCoV-OC43 samples and two HCoV-HKU1 samples. To test the infectivity of the clinical isolates, the HCoV-OC43 SGH-36/2014 clinical isolate and the HCoV-OC43 VR-1558 laboratory isolate were used to infect HCT8 colorectal cancer cells and Lifeline® human bronchial/tracheal epithelial (HBTE) cells grown in an air-liquid interface.

Interestingly, they found that SGH-36/2014 effectively infected HBTE cells and retained high replicative capacity, but did not infect HCT8 cells well. In contrast, the opposite was true for the laboratory strain. In HCT8 cells, VR-1558 had reduced infective capacity and could not replicate. However, VR-1558 efficiently infected HCT8 cells and retained replicative activity. These observations demonstrate that HCoV patient isolates function normally in a clinically relevant cell culture model, while the laboratory strain does not.

Next, to determine the route of entry of the virus isolates, the group treated HBTE air-liquid interface cultures with inhibitors of the cell surface TMPRSS2 protease or the endosomal protease cathepsin L. Compared to the VR-1558 laboratory strain, which was inhibited upon loss of cathepsin L activity, the infectivity of HCoV clinical isolates was largely inhibited by loss of TMPRSS2 activity.

The researchers next evaluated the expression of TMPRSS2 mRNA in HCT8 cells and found it was very low, suggesting that the low infectivity of clinical HCoV strains is likely due to reduced expression of its S protein activator. Interestingly, they did find that treatment of HCT8 cells with a cathepsin L inhibitor lowered both SGH-36/2014 and VR-1558 infectivity, suggesting that in the absence of TMPRSS2, clinical HCoV isolates used cathepsin L for entry.

Together, the results of this study identify important implications for HCoV research in that the clinical strains of HCoV-OC43 and HCoV-HKU1 do not use the same route of viral entry as laboratory strains and therefore, long-term use of lab strains may alter how these viruses behave in the clinic.

How are you using Lifeline® cells in you research? Let us know! If you follow our blog, you know that we are always looking for new research to highlight and new model systems to discuss!