An in vitro transepithelial migration assay to evaluate the role of neutrophils in Respiratory Syncytial Virus (RSV) induced epithelial damage

Yu Deng,Jenny A Herbert,Claire M Smith,Rosalind L Smyth

doi:10.1038/s41598-018-25167-4

Abstract

Large numbers of neutrophils migrate into the lungs of children with severe Respiratory Syncytial Virus (RSV) disease. It is unclear how these cells contribute to viral clearance and recovery from infection or whether they contribute to disease pathology. We have developed a novel in vitro model to study neutrophil migration through airway epithelial cells (AECs), the main cellular target of RSV infection. Our model reproduces a physiologically relevant cell polarity and directionality of neutrophil migration. Using this model, we found that RSV infected AECs induced rapid neutrophil transepithelial migration. We also detected increased AEC damage associated with RSV infection, with a further increase in epithelial cells shedding from the Transwell membrane following neutrophil migration. This was not observed in the mock infected controls. Neutrophils that migrated through the RSV infected AECs showed increased cell surface expression of CD11B and MPO compared to neutrophils that had not migrated. In conclusion, our in vitro co-culture assay can be used to identify critical mechanisms that mediate epithelial cell damage and promote inflammation in children with severe RSV disease.

Highlights

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation of infants in the developed world, causing bronchiolitis and severe lower respiratory tract disease in infants and young children[1]
We found that expression of CD11B, but not MPO, was significantly (P < 0.05) increased in neutrophils which had migrated through airway epithelial cells (AECs) infected with Respiratory Syncytial Virus (RSV) for 72 hour compared to non-migrated neutrophils after 1 hour (Fig. 5B)
We found that 24 hours post RSV infection, neutrophil migration led to a significant (P < 0.05) 48% reduction in the number of AECs present on the membrane compared to the mock control (Fig. 6C)

Summary

Introduction

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation of infants in the developed world, causing bronchiolitis and severe lower respiratory tract disease in infants and young children[1]. A fairly inexpensive in vitro model that enables the investigator to experimentally manipulate the viral infection, epithelial barrier, and/or neutrophil in a well-controlled, highly reproducible system is needed. This would be similar to that developed to model intestinal epithelial and lung epithelial responses to bacterial pathogens[14,15,16,17,18,19,20,21], which use cells lines to model the epithelial monolayer. More recently a model has been developed studying neutrophil migration during bacterial infection using primary airway epithelial cells[22]

Methods

Results

Conclusion