Experimental Virology - Ronald Dijkman

Host - pathogen interactions in respiratory epithelium

When a respiratory pathogen crosses the species barrier through inter-species transmission the pathogen can cause severe disease in the new host. This is well established for influenza A virus that is transmitted from its main natural reservoir, aquatic birds, to a wide spectrum of animal species, including poultry, swine, canine, equine and human and can have severe consequences on both animal and human health. The determinants affecting interspecies transmission or pathogenesis of emerging respiratory infections among different host species are only partially understood on the viral side, and there is still a great gap in our knowledge on cellular effectors and their mechanisms in the airway epithelium among the different host species. Thus, for both epizootic and zoonotic risk assessment and the development of effective counter measurements against these diseases it is necessary to define critical determinants of species barriers for emerging respiratory pathogens, such influenza viruses and coronaviruses.

The research of our group is focused on characterizing the (i) innate immune response in the respiratory epithelium, (ii) the influence of viral genetic traits in immune evasion and (iii) to define the influence of host determinants during respiratory virus infections.

To address these questions, we make use of a pseudo-stratified primary airway epithelial cell (AEC) culture system as main model. The AEC system is based on the isolation of primary lung epithelial cells that are manipulated with well-defined medium to form pseudo-stratified AECs. The pseudo-stratified AEC contains basal, secretory and ciliated cell populations, and generates mucus. Therefore, this in vitro system recapitulates many aspects of the in vivo airway epithelium, namely epithelial cellular differentiation and repair, the presence of well-defined cell types of the airway epithelium, and the physiological mucus barrier. Over the past years, we have established AEC models from a large repertoire of species, including human, porcine, bovine and bats, enabling us to characterize virus – host interaction in different host species (Figure 1).

Figure 1. Identification of MERS-CoV cell tropism. Expression of dipeptidyl-peptidase 4 (red), the functional MERS-CoV receptor on non-ciliated but not on ciliated cells (yellow) in primary AEC cultures (cover image: Nature, volume 495, number 7440, 2014).

More recently, we have made the AEC system amenable to genetic manipulation. Allowing us to both modulate and monitor the host response dynamics in the airway epithelium over time. Thereby this system serves as a paradigm to define critical determinants of species barriers in the airway epithelium among different host species (Figure 2).

Figure 2. Genetic modification of primary human AECs to monitor the innate immune response. A. Schematic layout of the lentiviral-based Mx1 reporter cassette that is incorporated into the host genome after lentiviral transduction. B. Monitoring of the IFN response kinetics in hAECs harbouring the Mx1-Gaussia luciferase reporter that were stimulated with 10 µg/ml of polyI:C via either the apical or basolateral surface. The secreted luciferase activity was monitored for 24 hours with 3-hour intervals. C. mRNA transcript level of endogenous MxA in comparison to the Gaussia luciferase reporter, after basolateral stimulation with 10 µg/ml of pI:C for 24 hours D. Fluorescent images showing the expression profile of the Mx1-eGFP reporter after basolateral stimulation with 10 µg/ml of polyI:C for 18 hours (unpublished data).