Exploring host-gut microbial genetic and immune interactions using twins

Microbes are crucial in maintaining a healthy immune system and are the link between our diet and health. We have around 100 trillion gut microbes that outnumber our human cells ten to one and our genes 100 to one. The microbes produce a wide range of enzymes, chemicals, hormones and vitamins that can potentially interact with our bodies. Alterations to our gut microbes have been implicated in many human diseases ranging from obesity, colitis, allergy and autism. Only recently thanks to genetic sequencing have we been able to study them properly and realise that the vast majority are not harmful to us and many are beneficial.

Study of the gut microbes is particularly exciting because we know that we can alter their composition through surgery, drugs, probiotics and diet. In this way we could potentially alter many diseases via microbial manipulation. We are currently experiencing an epidemic of allergies in the western world as well as increases in many autoimmune diseases and this could be related to abnormalities and recent changes in our gut microbes. The main method of communication between our microbes and our body is via the immune cells in the gut lining.

This is a study designed to provide vital information about the interactions between our human immune cells in our gut lining and the trillions of microbes that inhabit our gut. Recently we have discovered that the microbes found in our stools vary enormously between individuals and that some of this variation is due to differences in our human genes. However microbes in the stool do not necessarily represent the abundance or functions of the microbes in the gut wall, which may have even greater effect on host health. We lack vital information on the relationship between microbes in the gut wall and our host cellular responses which we need if we are to progress this exciting field.

This project will provide a valuable resource database of microbes in the gut wall and the cellular responses of the host in 100 pairs of identical twins. The benefit of using twins is that this allows us to determine effects which are independent of age, early family environment and importantly human genetics. We have recently shown that our own genes determine which species of bacteria proliferate in our guts which vary markedly between people.

Twin volunteers will be aged over 40 without any major diseases and will undergo a normal screening colonoscopy. We have already piloted our volunteers and found over 60% keen to participate. During the procedure we will obtain biopsies and brushings at 3 different sites in the bowel, going up to the end of the small intestine. This will allow us to test the different microbe species at each site and how the most important ones interact with the human immune genes (measured by levels of gene expression) and other markers of the immune system. As microbes rarely work alone as well as exploring the associated species we will look at the networks of microbes that have similar functions and build this into our resource.

The resource will enable other scientists in the field to look up potentially interesting genes and microbes and explore their respective functions. It will also allow scientists to compare microbe results from stools and predict the effects seen within the gut and the human immune system. Finally, we will do some pilot studies to test the effects of transplanting immune related microbes from humans into sterile mice which will test if the microbes are directly affecting the immune system. The ultimate aim will be to find the key microbes that help our immune system and develop ways to increase their numbers or their function through diet or other methods. The study has the potential to have enormous impact across many diseases and fields of medicine involving ageing, diet and the immune system. We have support letters from a wide range of academics across the world who would benefit from this work.

Human microbiome research is one of the most exciting areas of modern medicine given our ability to manipulate microbes. While studies have found microbial abnormalities in many traits and chronic diseases, these are often inconsistent in the type of microbes implicated. Limitations and biases of current approaches include the fact that case-control studies fail to account for genetic differences in microbiome populations which we have shown are important. Twins are an ideal way to account for genetics. In addition, virtually all studies rely on detecting microbes from the stool sample rather from the gut itself with major assumptions about the faecal sample matching the contents of the lower ileum and colon. Few studies have directly compared faecal and gut wall microbes in healthy humans. Understanding what influences the gut microbes at their site of interaction with the host immune system in normal people is crucial information that is currently lacking. We propose a unique twin study to quantify these changes and provide a resource of the gene-environment-immune relationships at 3 gut sites.

100 extensively genotyped and phenotyped postmenopausal MZ twin pairs aged 50+ will undergo a week's food diary, food frequency questionnaire, followed by stool sampling, bowel preparation and colonoscopy to the terminal ileum. brushings and faecal water will be sampled for 16SrRNA analysis at three sites in the distal gut (terminal ileum, ascending and descending colon). 18 colonic pinch biopsy samples divided equally between 3 sites will be split for RNA sequencing. In a subset of 10 twin pairs Treg cells will be separated and cell specific RNA sequenced to allow comparison and validation of intestinal and cell specific signals. Fresh blood samples will be separated into PBMC for immunophenotyping. We will also create a functional database of gene expression and microbial networks and finally perform a pilot study of causality of key immune microbes in germ free mice.

By the end of this three year grant - the data will be available and the benefits of the novel data will be accessible by all. Dissemination is an essential component of this project and right from the start, scientists will work on informing the public as well as other stakeholders about microbiome research which has the potential to have far reaching consequences for public health especially since intervention into, say, diet are non - invasive and a viable option to all.

Beneficiaries
Project participants: The staff working on this project will expand their analytical skills, become experts in the field and will be able to continue to develop microbiome research after the end life of this project as well as create a scientific network that will enable further research. Skills learnt will be applicable across all scientific domains.

Scientific community: This project will generate a knowledge base around the microbiome and host an open-access database to share findings and data with researchers working in all health domains. In addition, we will host an annual meeting about the microbiome to foster collaboration, to disseminate project results, to address any problems arising and to decide the future directions for microbiome research

Public: Knowing the composition of the gut microbiome will be of interest to the public at large in terms of health management.
Raising awareness of the interaction the gut bacteria have with the immune system and disease may encourage the public to get involved and possibly even participate in the British Gut Project where they can provide a stool sample and receive an analysis of their own gut microbiome.
Encouraging participation in this field will open the general public up to the scientific world which may initially seem daunting, thus benefiting scientists and public in opening dialogue for the long-term and facilitate participation in further projects in this field e.g. interventions.

Charities e.g. Crohns disease: A number of diseases have been proven to be associated with a disequilibrium in gut bacteria (not least Crohn's disease).
Throughout this project and beyond, we will interact with charities operating in relevant domains e.g. obesity so our research outcomes/results can be communicated to sufferers and clinicians leading to a better quality of life for those suffering a condition affected by gut bacteria.

Policy makers: The findings of this project will provide confidence in more widespread measuring of stool samples as markers of intestinal health and potentially change nutrition guidelines

Commercial sector: Findings of the research conducted in this project will be of great interest to the commercial sector. We have several links with commercial companies already but we will work to extend these links to other companies working extensively in the field of microbiome applications. These include
Crohn's Disease: Enterome BioScience, Coronando Biosciences and GT Biologics
C.difficile infections: Cipac Therapeutics, AvidBiotics (also E.coli), Symbiotic Health OpenBiome, Seres Health and Rebiotix
CardioVascular Disease: Cleveland Clinic
Colon cancer: ActoGeniX
Autoimmune, allergic and inflammatory disease: Symbiotix Biotherapies, Janssen Biotech
Inflammatory Bowel Disease: Second Genome and Janssen Biotech
Plaque psoriasis, autism, multiple sclerosis: Coronado Biosciences
Vaginosis inc Urinary Tract Infaction: Osel

Museums: Interactive displays on the gut bacterial composition may interest museums, particularly the Science Museum in London who we work with on a regular basis. Talks, interactive demonstrations and participation in specialised events can educate the public and further encourage involvement with science research.