Role of DNA methylation in regulating intestinal epithelial cell function in health and paediatric Inflammatory Bowel Diseases

Inflammatory Bowel Diseases (IBD) cause chronic inflammation of the large bowel (i.e. Ulcerative Colitis (UC)) or the entire intestine (i.e. Crohn's Disease (CD). It remains entirely unknown why the number of patients diagnosed with these conditions continues to increase so dramatically in recent decades, with up to 30% of patients being diagnosed during childhood, many now under the age of 10 years. In the absence of a cure, children are faced with lifelong disabling disease that impacts significantly on their growth and development. Moreover, the disease can also have a devastating effect on psychosocial development, not only for the child, but also for the entire family. Despite the major health burden caused by these conditions, our understanding of the causes and mechanisms of the disease remain limited.
It is agreed that malfunction of cells forming the inner lining of our intestine play a crucial role in causing IBD. These cells are called the "intestinal epithelium" and serve many important functions such as absorption of nutrients and water, as well as the containment of millions of bacteria, which play an essential role in our health and the digestion of our food. Therefore, the gut epithelium forms the crucial barrier between the environment and our body. Additionally, the epithelium can also activate specialised immune cells (called T-cells) for example when fighting gut infections. Although it is highly likely that impaired function of the gut epithelium contributes to the development of IBD, we know very little about the details of how this might happen in humans.
Many research studies have demonstrated that our environment may play an important role in causing diseases including IBD. However, how changes in our environment can cause impaired function of cells in our body is still unclear. The answer could be provided by a fundamental biological process called "epigenetics". This mechanism is able to regulate the function of cells in response to our environment. It could therefore provide a link between changes in our environment, increased appearance of IBD and specific changes in cellular function of effected tissues. In support of this hypothesis we have recently discovered epigenetic changes in the gut epithelium of children diagnosed with IBD. A gene called NLRG5 was found to be affected and is known to be involved in a pathway that is important in the interaction between the gut epithelium and specialised immune cells (i.e. T-cells). This interaction is also likely to be very important in IBD.
In this project we will now investigate how epigenetic mechanisms can change expression of NLRG5 in the human gut epithelium and what effect this has in children suffering from IBD. We will also study the communication between the gut epithelium and T-cells particularly with regards to potential changes in IBD patients.
All of the experiments we are proposing to do in order to answer our research questions, will be performed on human samples. We will analyse epithelium directly taken from children diagnosed with IBD and patients without any gut disease. Importantly, recent breakthrough discoveries have now made it possible to generate three- dimensional models of "mini - guts" in a dish, from only tiny human gut samples (biopsies). We have established these so-called "human intestinal organoids" in our laboratory and are now able to use them in this project. We believe that performing experiments on real human patient samples is very important as it dramatically increases the chances of findings having a direct impact on patient care including the development of novel drugs and treatment approaches.
A major strength of our project lies in the fact that doctors, scientists, other healthcare professionals, children and their parents are all working closely together. This collaboration provides us with unique access to a large number of human gut tissue samples.

Objective 1: Mucosal biopsies will be obtained from Terminal Ileum and Sigmoid Colon. Samples will be processed immediately for epithelial cell purification using magnetic bead sorting and generation of intestinal organoids according to established protocols. DNA and RNA will be extracted simultaneously. NLRC5 promoter DNA methylation analyses will be performed using bisulfite pyrosequencing and expression of NLRC5 as well as MHC-I related genes tested using RT-PCR and western blotting. Exposure of organoids to IFN-gamma will determine the impact of DNA methylation on the inducibility of MHC-I signaling. Locus specific, targeted de-methylation will be performed using a CRISPR/Cas9 based method and correlation between DNA methylation and clinical phenotype investigated using various computational approaches.

Objective 2: We will generate NLRC5 knock-out and over-expressing human gut organoids using CRISPR/Cas9 genome editing and piggyBac transposon-based vector system respectively. Organoids from healthy individuals obtained from 2 gut segments will be modified and co-cultured with IFN-gamma. Following, we will perform genome wide expression (RNAseq) and proteomic Tandem Mass Tag profiling on organoids. Generated data will be analysed using novel computational methods such as diffusion-based approaches, which will allow identification of NLRC5 and/or IFN-gamma dependant signalling pathways operative in the human intestinal epithelium.

Objective 3: In order to assess the functional impact of NLRC5 expression and MHC-I signaling on the intestinal epithelium in health and CD we will test intestinal epithelial barrier by measuring Transepithelial Electrical Resistance and efflux of FITC-dextran across organoids grown as monolayers. Additionally, we will test innate host response to viral infections and co-culture blood derived CD8+ T-cells with gut organoids and study their interaction using two-photon microscopy.

Patients suffering from IBD: Our ultimate aim is to improve the life of all patients and particularly children suffering from Inflammatory Bowel Diseases (IBD) as well as other related intestinal conditions (e.g. Necrotising Enterocolitis). The incidence of IBD has been rising dramatically in recent decades and patients seem to suffer an increasingly severe course of disease. In the absence of a cure and major lack of targeted, highly effective treatment options we urgently require novel approaches to improve our understanding of these conditions and develop improved strategies to develop better treatments. The proposed project has the potential to provide a substantial contribution towards these goals not only by directly generating novel knowledge, data and insight but also by further developing and validating a human cell-based model system, which will provide researchers with unprecedented opportunities to employ these tools in order to answer their own research questions.
Importantly, identification of cell type specific, molecular pathways that are altered in IBD provides an ideal starting point for the development of novel, targeted treatment approaches.

Medium to long term specific benefits of our work to patients suffering from IBD include the use of patient derived organoids to personalise treatment by testing efficacy of existing treatments, pre-clinical testing of promising new compounds and the development of entirely new drugs, which could specifically target the intestinal epithelium.

Socio-Economic impact: IBD affect approximately 1 in 250 people with currently 240,000 people diagnosed with condition in the UK. Associated healthcare costs to the NHS are an estimated £720 million per annum. In addition to calculated costs, the disease frequently has devastating psycho-social effects particularly on children, which frequently extend to their entire family. Hence, any improvement our work will have on their medical care is likely to have a wider effect on their family, friends and wider society.

Pharmaceutical industry: Human organoids have great potential to be used by the pharmaceutical industry. We have established active collaborations with pharmaceutical industry (i.e. MedImmune, Chimerix) which are based on the use of our human organoid model in IBD and viral infections. The prosed project will provide additional evidence for the use of organoids as translational research tools and will allow us to further develop the model thereby providing additional scope for future collaborations with industry.

Impact on academia and related research: We expect our work to be highly beneficial to a wide range of scientists, researchers and clinicians worldwide. These include those working within the field of IBD, epigenetics, stem cell biology and human organoid culture development. Importantly, genome wide datasets generated from highly purified primary human tissue samples (and matching organoids) obtained from a unique cohort of paediatric patients combined with the provision of detailed clinical information on all samples will enable researchers worldwide to address a wide range of novel questions by interrogating these data.