CO-REGULATION OF MACROPHAGE INFLAMMATORY PHENOTYPE BY IRF5 AND RELA

Inflammation is a normal and self-limiting physiological response to infection and injury but can lead to extensive tissue damage and disability when elicited in excess or sustained. Pathological consequences of chronic inflammatory responses include a variety of diseases with huge social impact ranging from autoimmune diseases, such as rheumatoid arthritis to many forms of cancer. Macrophages are cells of the immune system which patrol the body and have the ability to recognise a number of different signals released during infection and damage. They are of central importance in the pathogenesis of chronic inflammatory diseases. Depending on the type of signal they receive, macrophages can acquire "aggressive" inflammatory phenotype and will destroy invading threats and defective cells. They secrete inflammatory molecules and set up an environment for expansion of other types of immune cells with inflammation propagating features. Other signals give rise to more "peaceful" macrophages that promote the growth and repair of damaged regions. For example, synovial lesions in rheumatoid arthritis are characterised by the pre-dominant presence of aggressive macrophages.

Recently my laboratory has made a potentially therapeutically important discovery and identified a molecular switch, called 'IRF5' that controls the aggressive phenotype of macrophages: it is highly expressed in aggressive macrophages and induces a characteristic gene expression and inflammatory molecule secretion profile. It is crucially important for establishing an inflammatory environment, associated with a number of autoimmune and inflammatory diseases, and promotes development of memory immune cells which produce even more harmful inflammatory mediators. We have also explored modes of IRF5 regulatory function and demonstrated that they involve functional and physical interactions with another molecular switch, called NF-kB RelA, which is important for proper immune function of both aggressive and peaceful macrophages. This project will test the hypothesis that interfering with IRF5, and in particular with its interactions with RelA, will override signals influencing aggressive macrophage development at sites of inflammation and promote their transformation into more peaceful phenotype, thus reducing inflammation.

Our aim is to ascertain:

1. which inflammatory molecules produced by aggressive macrophages are regulated by IRF5 and RelA and how is this control achieved on the molecular level?

2. can we block direct interactions between IRF5 and RelA and would this reduce production of the inflammatory molecules identified above?

The outcome of this study is expected to be the proof-of-principle demonstration of a new approach to controlling sustained inflammatory cytokine production, based on modulation of the recently identified major molecular switch. The follow up studies will look into the possibilities of other ways of modulating its activity and function, using chemicals, amendable to drug development. Taking account the essential role of macrophages in any immune and inflammatory condition, identification of major molecular switches that determine aggressive or peaceful function of macrophages may hold the key to new therapeutic interventions. It is an important area of research, as it may result in new class of treatment for a wide range of immune conditions. For example, blocking the function of these switches would dampen down damaging inflammation present in autoimmune diseases, while alternatively, inducing their function would boost the immune system in people with immunosuppression.

Macrophages are immune cells that produce inflammatory mediators and are of central importance in the pathogenesis of chronic inflammatory diseases. The state of macrophage activation depends on the environmental factors and can change from pro- (M1) to anti-inflammatory (M2). M1 macrophages mediate resistance to pathogens and tissue destruction, whereas M2 macrophages promote tissue repair and remodelling as well as tumour progression. Mechanisms that control the switch of macrophage states present an attractive target for designing new therapeutic interventions that would modify sustained inflammatory macrophage activities without affecting their homeostatic function.

We have recently discovered that the transcription factor IRF5 is a major factor defining the pro-inflammatory M1 macrophage polarization. It directly regulates the secretion of specific inflammatory mediators characteristic of M1 macrophages (e.g. IL-12, IL-23, TNF, IL-1), that subsequently set up the environment for expansion of Th1/Th17 cells. We have also mapped molecular mechanisms of IRF5 function in regulation the TNF gene expression, such as direct binding to DNA and indirect recruitment via the formation of a protein complex with RelA. This proposal employs the state of the art functional genomics approaches (e.g. ChIP-Seq, RNA-Seq, EMSA-Seq) and established biochemical techniques (e.g. peptide mapping of protein-protein interaction interface) to identify a subset of genes important for M1 macrophage polarization and co-dependent on RelA-IRF5 interactions and to design molecules capable of breaking these interactions.

The proof of principle modulation of M1 macrophage polarization via interfering with RelA-IRF5 interactions may pave ways to future chemical drug design, which will selectively target the inflammatory response, ideally without having a deleterious effect on innate immunity which is an essential first line of defence against microbes.

With recently achieved understanding of the molecular mechanisms behind the set up of sustained inflammation, opportunities are now emerging to embark on a systematic and comprehensive analysis of this biological process and to develop new therapeutic strategies designed to control its magnitude and duration. As such, the proposed research could be advantageous for a wide range of beneficiaries, including industry and private sector; UK and EU policy-makers; patients with autoimmune conditions and charities supporting the research in inflammatory diseases; students and wider public interested in immune conditions.

1. Industry/private sector: The identification of amino acid groups involved in RelA-IRF5 interactions and the proof-of-principle modulation of specific gene expression by competing peptides provides a rational for a large-scale chemical library screen of compounds and/or drug design. This will benefit players in the industry and private sector, that are focused on chemical drug design and production, as they will be able to capitalise on our discovery and its implications furthered during the course of this project, by bringing about new drugs for chronic inflammatory and autoimmune diseases. We are currently engaged in exploring the possibilities of setting up a collaborative research programme with Novartis. Considering that the initial discovery and the research into ways of modulating IRF5 activity have been patented by the Imperial Innovations on our behalf, industry and private sector involvement will ultimately lead to fostering economic performance and competitiveness of the UK.

2. Policy-makers: The proposed research fully aligns with the MRC strategic priorities to train post-doctoral researchers in next-generation sequencing analyses while at the same time providing analytical capacity to UK-based experimental groups. The computational part of the proposal will be undertaking in close collaboration with CGAT, and thus it will contribute to training of computational biologists capable of analysing and interpreting next-generation sequencing data sets and will help to address the UK-wide shortage in this area. Our engagement in the EU scientific framework will ensure that the results of this project will be fully disseminated via the EU FP7 Model-In consortium web-site as well as communicated to the project officer, as the research which extends beyond the Model-In objective and together may shape the new FP7 funding calls.

3. Patients: IRF5, a molecule that determines whether key cells of the immune system promote inflammation or inhibit it, could hold the key to new treatments for autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. The proposed research together with already ongoing investigations in the physiological role of IRF5 in mouse models of inflammatory diseases, such as house dust mite induced asthma (grant application to the American Asthma Foundation), collagen induced inflammatory arthritis (grant application to the Arthritis Research UK), inflammatory bowel disease (fellowship application to the Wellcome Trust), obesity related inflammation has the potential to bring about new drugs and therapeutic interventions and thus to contribute to the nation's health and wealth.

4. Students and wider public: Our communication plan presents the opportunity for a wider dissemination of the results via taught under-graduate and post-graduate courses, such as Advanced Topics in Molecular and Cellular Immunobiology, co-designed by the principal applicant for the 3rd year Imperial College under-graduates in Biology/Biochemistry/Immunology. We have an excellent working relationship with the press offices of Imperial College and Oxford University, as well as the number of immunology review journals and news agencies and which widely publicised our discovery of the role of IRF5 in macrophage polarization. These will be used to publicised the results of this study.