A 3D air-liquid interface airway epithelial cell model to study pathogen interactions within the bovine respiratory tract

Aims

The aim of the project is to develop a 3D bovine airway epithelial cell model at an air–liquid interface that will enable the complex interactions of bacteria and viruses within the bovine respiratory tract to be investigated, without the use of live animals.

Background

Bovine respiratory disease (BRD) is a disease of cattle that involves complex interactions between different viral and bacterial pathogens. It causes significant economic losses to the cattle industry worldwide. Key to the development of vaccines and antimicrobials is a better understanding of the early events that take place when viruses and bacteria colonise the host respiratory tract. Methods that are currently available to do this are not well developed, and consequently a large number of experiments have to be done in cattle.

Research details and methods

Bronchi and trachea will be used from freshly slaughtered cattle at a local abattoir. Airway epithelial cells grown in submerged culture will be triggered to differentiate by creating an air-liquid interface. The epithelium will be biochemically, morphologically and functionally characterised to determine the optimum window for infection studies with a range of pathogens. Interactions between epithelial cells and pathogens will be assessed using quantitative and qualitative markers including expression of various cytokines. The aim is to provide a model that has the functional properties to replace in vivo studies.

The overall aim of the project is to develop a bovine airway epithelial cell (BAEC) model at air-liquid interface (ALI) to investigate interactions of pathogens at the bovine respiratory mucosa. This will be achieved through the following objectives: (1) characterize and optimize differentiated BAECS grown at ALI; (2) investigate interactions of M. haemolytica, BRSV and BHV-1 with BAECs; (3) investigate the proinflammatory innate immune response of BAECs to BRSV, BHV-1 and M. haemolytica; and (4) establish the relevance of the BAEC model to in vivo infection studies of young calves

Methodology. BAECs will be obtained from the bronchi or trachea of respiratory tract material derived from freshly slaughtered animals at a local abattoir. Airway cells will be grown to confluence on Millipore transwells as submerged cultures and differentiation will be triggered by creating an ALI. The integrity, structure and functioning of the epithelium will be monitored for 42 days by biochemical and morphological characterization to determine the optimal "window" for infection studies. Interactions of M. haemolytica, BRSV and BHV-1 with BAECs, both individually and in co-infection studies, will be followed quantitatively and qualitatively. Secretion of IL-1b, IL-6, IL-8, IL-10, IL-12, IFNg and TNFa will be measured to assess the proinflammatory innate immune response of the BAECs. The relevance of the BAEC model to the in vivo situation will be assessed by infection studies of young calves.

Scientific and medical opportunities. The development and characterization of this in vitro model will provide an invaluable tool for studying the interactions of bacteria and viruses within the respiratory tract of cattle without the in vivo use of animals. It will lead to further studies aimed at advancing our understanding of infectious disease within the respiratory tract of cattle that will have a major impact on the development of improved vaccines and antimicrobials.

Cozens D, Grahame E, Sutherland E, Taylor G, Berry CC, Davies RL (2018).  Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract. Sci Rep. 8(1): 853 doi:10.1038/s41598-017-19079-y  

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Project grant

Status:

Active

Principal investigator

Dr Robert Davies

Institution

University of Glasgow

Co-Investigator

Dr Catherine Berry
Dr Geraldine Taylor

Grant reference number

NC/L000822/1

Award date:

Mar 2014 - Nov 2017

Grant amount

£390,050