Development of an in vitro bioassay to assess the regulation of immune genes in nontraditional model species

Abstract

Three out of four emerging infectious diseases of humans originated in animals. The COVID‐19 pandemic highlights the importance of understanding how the immune systems of animals interact with viruses, bacteria, and parasites allowing them to serve as disease reservoirs. Unfortunately, the tools to study the immune system of most animal species are not at the level needed to investigate these interactions. Therefore, our laboratory is constructing a new reporter plasmid that will allow for the development of species‐specific transcription factor‐activated reporter cell lines. The initial proof of concept is focused on avian species. Birds are known reservoirs for zoonotic pathogens like influenza A viruses, West Nile virus, Salmonella, Campylobacter, among others. Understanding how the avian immune system responds to zoonotic pathogens is critical to understanding how they serve as reservoirs, and potentially developing novel ways to prevent disease in humans. Transcription factors regulating pro‐ and anti‐inflammatory genes were chosen and candidate binding sites conserved across multiple avian species were identified. These candidate transcriptional binding sites were inserted into a novel dual reporter plasmid and screened for their ability to control expression of green fluorescent protein. Plasmids containing validated binding sites will then be stably integrated into the genomes of different avian cell lines generating a transcription factor‐specific reporter cell line. These cell lines will then be used as a library to characterize the transcriptional response of various cell lineages following stimulation by different pathogens, pathogen pattern molecular patterns, and cytokines. Ultimately this platform will be expanded to other species, allowing us to compare the in vitro host response across different mammalian, avian, and teleost species. Shedding new light on the role the immune response plays in the pathogenicity of different zoonotic agents.