Pathogen interference describes the ability of a colonising pathogen to interfere with the ability of an incoming pathogen to establish infection. Bordetella bronchiseptica is a gram-negative bacterium that has been shown to block the ability of influenza virus to establish infection in the murine nasal cavity, although to date, the mechanism for this blockade is unknown. Here, we established ex vivo models of the nasal epithelia to examine host-pathogen interactions and elucidate the mechanism of influenza blockade.
The nasal epithelia is composed of distinct populations of respiratory and olfactory epithelial cells. Nasal respiratory and olfactory epithelia were harvested from C57BL/6 mice, each tissue type was expanded before differentiating at air-liquid-interface (ALI). At day 28 post-ALI, trans-epithelial electrical resistance demonstrated robust respiratory and olfactory epithelial barrier integrity. The presence of key epithelial cell populations and production of mucin in the nasal respiratory and olfactory ex vivo models was confirmed by confocal microscopy.
To elucidate the mechanism of B. bronchiseptica-mediated blockade of influenza infection, nasal respiratory and olfactory epithelial ALI cultures were inoculated with B. bronchiseptica for 30 hours prior to influenza infection. At 24 hours post-influenza infection, bacterial and virus titres were enumerated, RNA extracted for RNAseq analysis and monolayers fixed for to examine cellular architecture by histology and confocal microscopy. Using these ex vivo models, we demonstrate that B. bronchiseptica colonisation blocks influenza replication in nasal respiratory and olfactory epithelial cells, recapitulating previously described in vivo results. Confocal microscopy revealed an upregulation of mucins, a known inhibitor of influenza infection and a possible mechanism of influenza A virus blockade. RNAseq analysis of B. bronchiseptica-colonised and influenza infected nasal respiratory and olfactory cells is underway to provide further insights of influenza blockade in the nasal cavity.
Collectively, we have developed ex vivo models of the upper respiratory system to dissect host pathogen interactions and elucidate mechanism of B. bronchispetica-mediated blockade of influenza virus. Understanding the mechanism of influenza blockade has major implications for controlling influenza virus transmission events and can be used to develop countermeasures against respiratory viruses with pandemic potential.