The role of Mediator in T-bet-dependent gene activation and its dysregulation in mucosal inflammatory disease.

Our immune system functions to protect us from infection and keep commensal microorganisms that live on and within our bodies in check. However, the immune system can also become dysregulated, instead damaging organs and tissues. This can cause chronic inflammatory diseases, which include Crohn's disease, ulcerative colitis and celiac disease, which primarily affect the bowel. These diseases cluster in families, suggesting a genetic component. Unfortunately, the number of people affected by these diseases is increasing and the treatment options that are currently available are poor.

There are a number of components of the immune system. CD4 T cells secrete signals that help other cells control microorganism numbers. CD4 T cells are normally resting but when the cell detects a foreign object, the cell changes into one of a number of different types of effector cell, each of which activates a different component of the immune response. The type of effector cell it becomes depends on the type of foreign object detected and allows the immune response to tailor itself to different threats. It is also important that the appropriate balance between these different effector types is maintained because excessive activation can lead to inflammatory diseases.

The differentiation of CD4 T cells into effector lineages is controlled by transcription factors, proteins that bind to specific sites on DNA and turn on and off near-by genes. Thus, if we could understand how these transcription factors function to turn on and off genes, and how this changes in disease, we might be able to develop drugs that modulate this process and block damaging immune responses.

We have identified a set of proteins that are required by a transcription factor called T-bet to activate immune response genes. Blocking the activity of one of these proteins, Cdk9, reduced inflammation in a model of the inflammatory eye disease, uveitis. We have also found that genetic variants that are more often found in people with inflammatory bowel disease affect the ability of T-bet to bind to DNA. This suggests that these genetic variants increase disease risk because they obstruct how immune genes are normally turned on and off.

The aim of this project is to determine how T-bet works with the proteins we have identified to turn immune genes on, to discover how the genetic variants associated with disease affect this process, and to test whether blocking the function of Cdk9 and other proteins in this pathway can reduce symptoms in models of inflammatory bowel disease.

This work will increase our understanding of the normal process through which genes are turned on during an immune response, which will be important to support this process in people whose immune systems are not functioning well. This research will also reveal how the genetic variants present in some people cause dysregulation of the immune system and how this can increase the risk of those individuals developing inflammatory diseases. Finally, this work will identify a set of possible drug targets through which chronic inflammatory diseases might be treated in the future.

Crohn's disease (CD), ulcerative colitis (UC), and celiac disease, are associated with dysregulated Th1 responses. These conditions have a genetic element, with many disease variants lying in non-coding regions. Disease incidence is increasing and current treatment options are poor. Thus, there is a pressing need for new insight into the molecular mechanisms controlling Th1 cell differentiation and its dysregulation in inflammatory disease.

Th1 cell differentiation is controlled by the transcription factor T-bet. We have recently demonstrated that T-bet functions to recruit Mediator and the super elongation complex to activate Th1 genes. Furthermore, this pathway renders Th1 genes hypersensitive to CDK9 inhibition. We have also found that T-bet binding sites are highly enriched at disease-associated SNPs and that disease variants alter the normal pattern of T-bet binding in cells.

We propose to determine how T-bet functions with Mediator to activate Th1 genes, establish how disease-associated polymorphisms affect these mechanisms, and test whether this pathway is therapeutically tractable for the treatment of mucosal inflammatory disease. This multi-disciplinary research project combines the expertise of the Jenner and Lord labs and a set of expert collaborators. We will dissect the role of T-bet and Mediator using 4C-seq, ChIP-seq and proteomics in conditional mouse knockout lines. Changes in T-bet and Mediator binding at disease-associated SNPs will be assessed by ChIP-seq in cells from donors of defined genotypes. The efficacy of CDK9 and CDK8 inhibitors will be tested in a mouse model of colitis and in explants from UC and CD patients.

The work will define the molecular mechanisms controlling lineage-specific gene expression and its dyregulation in inflammatory disease and reveal whether this pathway is therapeutically tractable. The results will have important implications for the regulation of other immune-mediated disease processes.

Please also see the attached Pathways to Impact.

Fit with MRC Strategic Priorities: The proposal provides an excellent fit with Theme 1: Resilience, repair and replacement, especially Natural protection and Tissue disease and degeneration, which focuses on chronic inflammatory conditions. Our proposed work also fits well with Theme 2: Living a long and healthy life, especially Molecular datasets and disease. Specifically, this proposal seeks to employ an integrative approach to determine the role of specific transcriptional pathways underlying T helper cell differentiation and its dysregulation in disease, and to determine whether these pathways may be therapeutically tractable. These objectives are aided by the use of a model system that closely phenocopies human IBD, combined with IBD patient samples.

Public sector: The identification of a new therapeutically tractable pathway for the control of auto-inflammatory mucosal disease could have a huge impact across the NHS and the social care sector. The incidence of IBD and celiac disease is rising, with an annual cost to the NHS of >£700M. Current treatments options are poor, with sub-optimal or non-sustained responses and side effects attributable to treatment. Medication failure is also common, with 44% of UC patients requiring surgical resection by 9 years. We are well placed to translate mechanistic findings from this project into relevant impacts as this project is taking place in the context of both the UCLH and Guy's and St Thomas' Biomedical Research Centres. This will ensure that useful discoveries are rapidly translated for maximum societal benefit.

Industry: The treatment of autoimmune and inflammatory conditions is in the top three priority areas of most big pharmaceutical companies due to both the therapeutic tractability and enormous market size. There is also a great deal of current interest in targeting transcriptional regulators. Therefore the potential for economic impact is very high. A number of CDK9 and CDK8 inhibitors have recently been developed and have entered clinical trails for cancer. BET domain inhibitors, which also act to block CDK9 recruitment to genes, have also been developed and are in clinical trails for cancer. We have previously demonstrated the efficacy of the CDK9 inhibitor flavopiridol in a mouse model of uveitis. There will be great interest if our work demonstrates that CDK9 and/or CDK8 inhibitors also show efficacy for IBD and celiac disease. Our research may also provide impetus for the development of other CDK9 and CDK8 inhibitors, or inhibitors of other aspects of T-bet, Mediator and super elongation complex function.

General public and societal impact: This work will stimulate the development of therapeutic agents for chronic inflammatory diseases such as IBD. IBD preferentially affects young, economically active patients, who experience significantly more lost work days and more unemployment than unaffected people. By identifying the mechanisms underlying Th1 cell differentiation, this work may also lead to interventions that would enhance T cell lineage specification to support immunity and vaccination. Determining the molecular basis of the association of particular genetic variants with disease risk will increase awareness in the general population for how interplay between genetics and the environment underlies our health and wellbeing.

Skills development: This grant will directly lead to the training of two staff members and provide a springboard for Dr Hertweck to follow previous postdocs in the lab and transition to an independent position. Dr Hertweck will gain expertise in 4C-seq, proteomics and bioinformatics that will expand his skill set and that of the UK as these advanced methods are propagated through the workforce. Staff will also gain training in skills transferable to the wider economy, including time management, communication, presentation, IT, computer programming and university teaching.