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Title: Developing three-dimensional co-cultures of the small intestinal epithelium with intestinal dendritic cells as a disease model for enteric infections
Author: Johnston, L.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Current models for studying enteric infections fail to fully recapitulate the complexities of the small intestinal epithelium in vivo and are also too simplistic in that they do not contain elements of the mucosal immune system such as dendritic cells, important in intestinal homeostasis. More complex models incorporating these elements would be beneficial to study enteric infections, such as T. gondii, which enters the host through the small intestinal epithelium and can cause severe encephalitis and birth defects. Identification of the host and parasite pathways involved in invasion of the epithelium by T. gondii might lead to the generation of novel therapeutics or vaccines. Organoid cultures have recently been developed, where intestinal stem cells divide in vitro to generate 3D structures recapitulating the cellular diversity and architecture of the small intestinal epithelium. This makes organoids a promising model for studying enteric infections and inflammatory diseases. However, organoids have not been fully exploited for studying enteric infections, due to an inaccessible luminal surface. Furthermore, very few studies have attempted to co-culture immune cell populations with the organoids. The work carried out in this doctoral thesis set out to address the lack of suitable models for studying early infection events. Organoids were dissociated to expose the luminal surface to infection by T. gondii. Live imaging showed the expulsion of infected cells into the lumen, a possible mechanism for the spread of infection in the intestine seen in vivo. The injection of parasite effector proteins into host cells that were not subsequently infected was observed, suggesting an extensive manipulation of host cells. This method will allow for high throughput analyses that could be used in drug screenings for the development of novel compounds that protect against infection. However, infection events are not restricted to the luminal surface of the epithelium, the physiologically relevant route of infection. To address this, protocols were developed for the microinjection of T. gondii into the luminal space of organoids. This method restricts infection to the luminal surface and allows for the visualisation of parasite infection, transmigration, and host cell responses. Dendritic cells with a small intestinal phenotype were derived from bone marrow, providing a widely accessible method for studying the function of intestinal dendritic cells. Co-culture of these dendritic cells with organoids provides a novel model that captures more elements of the intestinal environment than existing models. Co-cultures led to the unexpected finding that organoids suppressed aldehyde dehydrogenase expression in dendritic cells, while live imaging revealed characteristic behaviours such as luminal sampling, and surveillance of the organoid surface by dendritic cells. The models developed in this study provide important tools in studying host-pathogen interactions during early infections of the small intestinal epithelium. They can be adapted to include organoids and dendritic cells derived from knockout or transgenic mice that may elucidate the molecular pathways involved in intestinal homeostasis. These can also be applied to a wider field of research including inflammatory diseases, such as Crohn's disease, whereby dendritic cells are suggested to exhibit abnormal responses to the commensal flora. Improving our understanding of the mechanisms involved in intestinal homeostasis and infection using these novel models can lead to the generation of novel therapeutics and vaccines.
Supervisor: Coombes, Janine Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral