Identification and characterisation of causal molecules for Crohn's disease and ulcerative colitis

Summary
Crohn's disease (CD) and ulcerative colitis (UC) are the two major inflammatory bowel diseases. Both conditions occur in about 1 in 1000 of the population and have a major impact on those affected. Up to 75% of CD and 25% of UC patients will require surgery at least once, and both conditions are associated with a significantly increased incidence of gastrointestinal cancer.
Because they generally start in the second decade of life, and are usually lifelong conditions, they have a major detrimental effect on the social, professional and economic life of the affected individual. Direct costs of these diseases to the NHS are approximately 1 billion pounds per year.
The methods used to achieve a diagnosis of these conditions are imprecise, and largely descriptive. They consist of X-rays, colonoscopy, and as a more recent development, an imaging capsule can be swallowed. Small bits of the lining of the bowel can be removed for histological examination, which produces a subjective assessment of the microscopic appearance.
The development of new molecular technologies has created the possibility of identifying the basic mechanisms causing these diseases, and the molecules responsible, which could lead to more accurate diagnosis and the development of more effective treatment.
We have established that:
These two diseases are very different. CD is an immunodeficiency in which the patient's early inflammatory response to bacteria entering the tissues is ineffectual, leading to defective removal of bacteria from the tissues, resulting in chronic inflammation. By contrast, inflammation in UC is abnormally strong. CD affects the large and small bowel and the inflammation penetrates deep into the tissues whereas UC is confined to the superficial lining of the large bowel.
In CD the macrophage, a conductor of the immunological orchestra, is defective in a large proportion of patients leading to impaired inflammation. To identify the reason for this we collected macrophages from patients and healthy control subjects and found several genes to be expressed at abnormally low levels in patients. So far we have tested two of these, ADAMDEC1, an enzyme that chops up proteins, and Optineurin, a linker molecule that is involved in moving proteins inside cells. We developed fish and mice that were lacking either ADAMDEC1 or Optineurin in order to investigate their role in bowel inflammation, and the absence of either gene caused an increased inflammation in the bowels of the animals, consistent with the effect observed in patients.
We applied a similar approach to investigating the cause of UC. In this case we examined the pattern of expression of genes in the lining of the bowel, because of prior research indicating that this lining was abnormally friable in these patients. We discovered two genes that were translated at abnormally low levels in significant numbers of patients. One, Claudin 8, is involved in holding the lining cells together, and virtually nothing is known about the other, FAM5C. We wish to perform a similar study on bowel samples from patients with UC from Iceland, where the disease is very common and there is a small gene pool.
We will characterise these four proteins in order to better understand their function and to determine how they predispose to the development of IBD. We would also like to investigate some of the other strong candidate genes that we have identified.
IBD associated molecules might be very useful in molecular diagnostic tests.
Finally we will attempt to correct the expression of target genes in either the macrophage or bowel by using drugs and/or gene replacement therapy, with the hope of identifying targeted IBD therapies.
Our research programme is designed to identify and characterise molecules and mechanism responsible for the development of IBD, which will lead to improved diagnosis and treatment of these conditions.

The aetiology and pathophysiology of CD and UC are different. There is evidence of an innate immune deficiency in CD and a mucosal abnormality combined with accentuated acute inflammation in UC. To identify potential causal molecules we studied microarray data collected from macrophages and from mucosal biopsies from CD and UC patients. We identified a number of abnormally expressed genes in macrophages from CD patients, and went on to demonstrate that two of these molecules, ADAMDEC1 and Optineurin have novel roles in mucosal immunity. We plan to screen other under-expressed molecules in a zebrafish bowel inflammation model, and if they demonstrate a strong phenotype we will investigate their function in knockout (KO) mice. These molecules will be examined for an epistatic interaction with NOD2 in double KO mice.
Expression studies on the mucosa of UC patients revealed that Claudin 8 and FAM5C are grossly under-expressed in significant proportions of patients. FAM5C has never previously been associated with UC. We will perform expression studies on bowel biopsies patients from Iceland where there is a very high incidence of UC and a relatively small gene pool. Claudin 8 and FAM5C and candidate molecules from Iceland will be screened in zebrafish and those with a strong phenotype will be investigated in KO mice.
We will investigate the cell biology and biochemistry of ADAMDEC1, Optineurin, Claudin 8, FAM5C and other strong candidates to establish the mechanistic basis upon which deficiencies predispose to IBD.
Patterns of under expression of genes like FAM5C in the bowel mucosa might provide the basis for molecular diagnostic tests for UC. We are collaborating with Epsilon-3 Bio Ltd, who are developing drugs to enhance the immune response, and Phil Crosier who has a library of drugs licenced for other purposes that protect zebrafish against bowel damage, and will test their drugs in our mouse models with a view to using them therapeutically.

1. Beneficaries
A. Crohn's disease and ulcerative colitis (UC) cause a massive economic and human cost worldwide. In the UK these diseases affect one in 500 of the population with an annual economic cost of approximately £1Bn. These diseases generally affect individuals in the second decade with serious morbidity and profound and long lasting social and indirect economic costs. Conventional treatment has been inadequate. The newer anti-TNF drugs are very expensive, are only efficacious in about a half of the patients and a significant proportion of these become refractory to treatment. Effective oral therapy evidently represents an urgent unmet need in contemporary medicine.
Both these conditions predispose to a greatly increased incidence of bowel cancer, the causal mechanism of which is unknown.
Current diagnosis of IBD is dependent upon clinical, morphological and histopathological investigations, and the move to the establishment of diagnostic criteria on the basis of molecular pathology would be a major advance.
The demonstration that Crohn's disease can be caused by an initiating immunodeficiency will provide a pointer to pathogenic mechanisms to be investigated in other granulomatous conditions.
It is possible that colonic bowel disease will provide a good clinical setting for the development of gene therapy directed to the replacement or upregulation of molecules we identify as defective in colonic Crohn's or UC.
All of the data generated in this project will be available to download through online data repositories after publication. The large amount of transcriptomic data from extremely well characterised patients will be of particular interest to specialists in IBD as well as statistical bioinformaticians. We will also work closely with consortia working on IBD and transcriptomic data and, when needed, supply additional information. We currently have an on-going collaboration with the international IBD consortium.
B. Patients will ultimately benefit from more accurate diagnosis and rational treatment. Current treatment of Crohn's suppresses the symptoms, but this immunosuppression probably compounds the pathogenic problem, turning a sporadic into a chronic condition.
C. Public sector, business and industry. The applications of the proposed work will apply directly to the diagnosis and management of IBD, which is ineffective in these conditions. The molecules we identify might form the basis for chip based diagnostic arrays. For conditions such as this, the pharmaceutical industry has largely concentrated upon immunosuppressants. Immunostimulants that enhance macrophage function might be useful in Crohn's disease to maintain a remission. This has been confirmed in principle with previous promising compounds, which were unfortunately not clinically useful (Levamisole, Segal AW et al Br Med J. 1977 Aug 27;2(6086):555).
We have set up collaborations with Epsilon-3 Bio Ltd, Phil Crosier and Adrian Thrasher with the aim of identifying the most effective way of elevating gene expression levels of the molecules demonstrated to be abnormal in IBD patients. This approach will hopefully identify novel therapies which provide protection against the development of bowel inflammation. Positive findings will be of significant financial and commercial interest. We are thus ideally placed for translational development of our findings.
D. General public engagement. The project gives an important exemplar of how the development of experimental models for understanding the disease mechanisms can lead to new therapeutic approaches, emphasizing the importance of evidence based medicine in health care.
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