Mechanisms of intestinal epithelial responses to inflammatory modulators

The intestinal tract is a major barrier between the outside world and the body. It is responsible for digesting food and absorbing nutrients from ingested material. To aid digestion, the gut contains a large number of bacteria that are necessary to break down food so nutrients can be absorbed. The lining of the gut also secretes a host of chemicals that further support digestion. In addition, because the intestinal tract is directly connected to the outside world it is continuously exposed to viral and fungal species. To protect the body against invasion of all these potentially pathogenic factors, the gut wall is associated with an extremely complex system of immune surveillance. Although developed to protect the body against infection, this system can become deregulated and cause devastating inflammatory diseases of the small and large intestine including Crohn's disease, ulcerative colitis and inflammatory bowel disease. Furthermore, in cancer of the bowel, increases in markers of inflammation are associated with poorer prognosis. On the other hand, the ability of immune cells to regulate the growth and movement of cells in tissues that form tumours, make them an attractive force to harness in fighting cancer. To this end, a detailed understanding of the communication between immune cells and normal and cancerous tissues is required to (1) treat and diagnose inflammatory diseases of the intestinal tract and (2) utilize immune cells for treating tumours. To date, studies aimed at understanding how tissues and immune cell affect and regulate each other were limited to using either isolated cells or whole animals. Both approaches have drawbacks: the former involves non-physiological conditions that do not reflect the normal cellular environment of whole tissue and thus can not recapitulate the complexity of tissue organization normally found in the body; the latter does not lend itself to observing tissue and cell dynamics and also requires continuous sacrifice of animals (mice). Our proposed work utilises tissue intestinal organoids or 'mini-guts grown in a dish' that recapitulate normal intestinal tissue organization but can be manipulated and observed outwith the animal. Moreover, these organoids can be propagated in culture for long periods and they can be frozen and stored for future use. This greatly reduces the number of animals required for experiments. We propose to use intestinal tissue organoids to determine how immune cells and the intestinal lining interact and regulate each other. Specifically, we will measure how tissue and cellular organization and behaviour are affected by specific factors that are produced and secreted by different types of immune cells normally found in intestinal tissue. We will also examine how genetic changes in tissue that are known to affect immune responses affect tissue organization and function to understand how disease states associated with particular mutations predispose to inflammatory intestinal diseases. As part of this work we aim to establish a tissue bank of organoids that contain disease-associated mutations or markers that can report on the cellular processes in the context of tissue.
The outcome of this work will be an increased understanding of how the immune system can affect normal intestinal tissue and function and how it produces changes associated with inflammatory diseases. Furthermore, establishing a bank of well-characterized organoids for use of the entire scientific community will greatly accelerate our ability to generate novel and comprehensive data and also establish the potential for drug screening without the use of animals.

Inflammatory bowel diseases (IBD) are on the rise in the western world, and colon cancer is a leading cause of cancer-related deaths worldwide. Both these diseases involve the intestinal epithelium, the single layer of epithelial cells that is responsible for nutrient absorption and also for protection from the external environment. Associated with the gut epithelium is a complex network of immune cells that help to protect the epithelium against environmental insults. The continuous exposure to food-derived particles, innocuous microbial products from commensal bacteria, as well as pathogenic bacteria, viruses and fungi, require the gut immune system to maintain a fine balance between non-responsiveness and full activation and inflammation. Understanding how the epithelium responds to inflammation is of paramount importance to understand how the epithelium contributes to resolution of inflammation. Furthermore, knowing the mechanisms that contribute to defects in this balance as occurs in IBD is key to detect and treat these common diseases. Using intestinal epithelial organoids our work aims to determine the molecular detail that underpin responses of the epithelium to different inflammatory mediators, specifically how they affect proliferation, differentiation, gene expression, and barrier function. Furthermore, we will identify signalling pathways involved and measure the contributions of novel immune response modulators including the aryl hydrocarbon receptor (AHR) and Hypoxia-inducible factor 1 (HIF1alpha). This will establish how inflammatory responses in the gut are coordinated. Finally, we aim to create a pilot repository of intestinal organoids derived from genetically modified mice that will serve as an experimental resource for animal replacement within the scientific community.

This project will significantly reduce the number of animals used in experimentation by replacing whole animals with cultured organoids that can be regenerated in culture. The types of experiments we are proposing would normally involve 10-20 animals per week and would involve injection and oral gavage of animals with cytokines and activators to induce recombination of transgene. For the experiments in this proposal establishing organoids from each genotype will require 3 mice at most so the total will be 30 or less a year, including breeding and maintenance. Using organoids completely avoids having to treat animals with tamoxifen or other inducers of recombination because in organoids, the same activation can be carried out in vitro. Although, the treatment itself is usually not harmful to the animal, the consequences of the genetic changes they produce can be. Additionally, the use of organoids eliminates the need to develop tissue-specific reporters or conditional knockout mice, as the epithelia can be studied independent of other cell types. Thus new mouse strains will not be generated.
Laboratories all over the worldwide study gut and other epithelia and we anticipate that once a robust correlation between organoid responses and responses in vivo is established the number of mice needed will be reduced by thousands. Given the importance of inflammation in the gut and the correlation between inflammation and cancer, it is highly likely that our study will provide insights into how organoids can be used for drug screening to take into account the interactions between the immune system and epithelia. Drug screens still require Xenografts although their usefulness is questionable. Work in our proposal will establish whether organoids can report accurately on the interaction between the immune system and epithelia.
Thus establishing organoids as a routine system would reduce the suffering of animals significantly.