Apical-out chicken enteroids with leukocyte component as a model to study host-pathogen interactions

Abstract

Over the last 20 years global poultry production has tripled with approximately 107 million tonnes of chicken meat and 1.3 trillion eggs now produced every year (FAOSTAT, 2019). Of particular concern for all types of poultry production systems are zoonotic infections such as avian influenza virus and salmonellae, but also protozoal parasites like Eimeria which have a significant impact on animal welfare and the economy. Most of these infectious agents enter the chicken via the respiratory or intestinal epithelium, however a detailed understanding of how certain pathogens infect the gastrointestinal tract and airways in the chicken is lacking. In vitro avian gastrointestinal studies have long been hampered by a lack of representative cell culture tools. Mammalian 3D enteroids mirror in vivo intestinal organisation and are powerful models to investigate host-pathogen interactions, food science and improved targeting and efficacy of pharmaceuticals. The main aim of this study was to develop and characterise an in vitro 3D enteroid culture system from the intestine of chickens which would recapitulate the in vivo gut epithelial cell content, architecture and function. Once these cultures were established the aim was to determine whether the enteroids could be used to study the interaction of various pathogens with the enteroid epithelia, and to explore induced mucosal immune responses. Throughout these studies the characteristics and responses of the chicken enteroids were to be compared to those of the in vivo chicken intestine.

From this work, protocols to successfully isolate stem-cell containing villi or crypts from the embryonic and mature poultry intestine and culture multi-budding enteroids have been developed. These 3D intestinal models are shown to mature rapidly within 1-2 days and re-create the 3D villus-like architecture. Methods used to validate the enteroid cultures show they remained viable for over 7 days. Histological, transcriptional and functional analyses demonstrate the cellular diversity, barrier and digestive functions of the in vivo chicken intestinal epithelium in this chicken enteroid model. Unusually, the poultry enteroids develop rapidly and optimally in suspension without the structural support required to produce typical mammalian enteroids, resulting in an apical-out conformation with media-facing microvilli. Another striking feature of this model is the innate presence of functional intraepithelial and lamina propria leukocytes in a lamina propria core. Specifically NK cells, mononuclear phagocytes, and T cells have been identified, and phagocytosis studies have evidenced their functionality. Other lamina propria cell types have also been identified, revealing further complexity to this comprehensive in vitro model. To expand the potential applications of these cultures, enteroids were developed from two species of poultry and from different regions of the small and large intestine, which reflect in vivo intestinal architectural characteristics. In-depth RNA seq analysis has confirmed the reproducibility and consistency of these cultures as well as further exploring their digestive and immune functions, cell content and stage of development in culture. Analysis of stem cell growth factor transcripts indicate enteroid differentiation and homeostasis is predominantly maintained by cell populations within the enteroids. The transcriptome analysis also indicated the difficulties in passaging this culture system may be caused in part by a combination of epithelial-mesenchymal transition and deficiencies in a small number of downregulated growth factors. Methods to cryopreserve both villi and enteroids have therefore been developed to reduce the requirement for fresh tissues for every experiment and enable the sharing of material between labs.

The rapid maturation and advantageous polarisation of these 3D cultures enabled active infection of the epithelial apical surface with the bacterium Salmonella Typhimurium, influenza A virus as well as the avian-specific protozoan Eimeria tenella without the need for complicated manipulation. Multiplexed RT-qPCR Fluidigm Arrays supported analysis of the enteroid immune response to infection, demonstrating the differential regulation of an array of genes which mirror in vivo studies. In addition, permeability assays to analyse the enteroid epithelial barrier were developed and revealed significant differences in intercellular junction disruption between invasive and T3SS1 mutant S. Typhimurium infections.

The paucity of representative poultry gut in vitro models has limited research in this essential field of gut health and led to an over reliance on the use of live birds for experiments. This study has developed protocols to successfully isolate villi and crypts from the avian small intestine and derive 3D enteroids with an accessible epithelial layer. These rapidly maturing, highly reproducible cultures have been demonstrated to contain a comprehensive array of functional small intestinal and lamina propria cell types. The unique conformation in combination with the lamina propria component of the enteroids means they can be readily applied to study complex epithelial and leukocyte responses to bacterial, viral and protozoal pathogen challenge. The ability to biobank stem cell containing tissue alongside the demonstrated physiological relevance of this model, means the floating chicken enteroids satisfy many of the guiding principles of the 3R’s which are being increasingly implemented in new legislation. In summary, this work has created a valuable analytical tool which enables a detailed view of how the intestines of the post-hatch chick grow and respond to external challenge in vitro. This model has the potential for use in host-pathogen, pharmaceutical and food science research as well as commercial applications for e.g. probiotic and vaccine batch testing.