NIHRDataInsights/HRCSData huggingface dataset:
Title: The interplay between protein phosphorylation and ubiquitylation in regulating the signalling networks of the innate immune system
Funder: Medical Research Council

Abstract: The innate immune system is vital for defence against infection by microbial pathogens, such as bacteria and viruses, but the overproduction of inflammatory mediators, or the failure to resolve inflammation once it has served its purpose, causes many inflammatory and autoimmune diseases. They include arthritis, asthma, colitis, lupus, psoriasis and sepsis and even blood cancers, such as lymphomas. An understanding of the signalling networks that control the production of inflammatory mediators at a molecular level is therefore critical to identify key targets for the development of drugs to boost resistance to infection or to suppress inflammatory and autoimmune diseases. Protein phosphorylation and ubiquitylation are the two key post-translational modifications that are deployed to regulate the innate immune system. The MRC supported research in my laboratory is focused on understanding how the key transcription factor NF-B and Interferon Regulatory Factors (IRFs) are switched on to control the production of pro-inflammatory cytokines and interferon (IFN). Over the past two years significant advances in understanding how NF-B and the IRFs are regulated have been made by the three Ph.D. students in my lab, who are supported by MRC Unit funding. My student Sam Strickson has made important contributions towards the discovery that a hybrid ubiquitin chain, comprising both Lys63-linked and Met1-linked ubiquitin oligomers, is critical to switch on the IB kinases (IKKs) that activate NF-B and that the formation of Lys63-linked ubiquitin chains is a prerequisite for the formation of Met1-linked ubiquitin chains. Sam discovered that the anti-inflammatory drug BAY 11-7082 blocks the activation of the IKKs by inactivating the E2 conjugating enzymes required for the formation of Lys63-linked and Met1-linked ubiquitin oligomers. Excitingly, he also found that this drug is effective in destroying cancerous cells from patients with Diffuse Large B-Cell Lymphoma, one of the least treatable forms of this blood cancer. My student Jiang Zhang has discovered that the activation of the IKKs requires two different protein kinases that phosphorylate distinct serine residues in the “activation loops” of the IKKs, a control mechanism that may be of general significance. Another student Nazma Malik has made the unexpected discovery that a drug developed as an inhibitor of the protein kinase PLK1 for the treatment of cancer, blocks the production of IFN during viral infection by a novel mechanism that is independent of PLK1 and which prevents the recruitment of RNA polymerase II to the IFN gene promoter. This drug and other molecules with a similar mechanism of action may have therapeutic potential for treating the lethal effects or flu and/or sepsis in which the over-production of IFN is a major contributory factor.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The Role of GIMAP GTPases in Lymphocyte Homeostasis
Funder: Medical Research Council

Abstract: The mammalian GIMAP GTPases are implicated in the regulation of lymphocyte survival. In rodents, mutations in two members of this family cause severe lymphopenia in either the T lineage alone (GIMAP5) or in both T and B cells (GIMAP1). This application proposes a multidisciplinary programme of work aimed at understanding the mechanism of action of these GTPases and the extent of requirements for them in the function of mature lymphocytes. Two proposals to explain the essential pro-survival properties of GIMAP1 & 5 will be addressed (Aim 1). These are (i) their modulation of the intrinsic apoptotic pathway by interacting directly with Bcl-2 family proteins and thereby influencing mitochondrial outer membrane permeabilisation and (ii)their participation in autophagic mechanisms required for the development of mature lymphocytes from immature precursors (thymocytes; bone marrow B cells). In the latter case, particular focus will be applied to the regulated autophagic control of mitochondria, termed mitophagy, the importance of which in T cell development has recently been uncovered. The work will also ask (Aim 2) whether the requirement for GIMAPs 1 & 5 extends to long term lymphocyte survival in vivo and to the generation and maintenance of antigen-specific memory cells. It will also assess a possible role for GIMAP GTPases in T helper cell differentiation. These investigations will make use of a mouse strain bearing a conditional allele of GIMAP1 that, when combined with tissue- or inducer-dependent Cre recombinase alleles, will allow us to delete this gene selectively in lymphocyte lineages in vivo or inducibly either in vivo or in tissue culture.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: CO-REGULATION OF MACROPHAGE INFLAMMATORY PHENOTYPE BY IRF5 AND RELA
Funder: Medical Research Council

Abstract: 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.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Systems approach to the analysis of T cell receptor signal transduction
Funder: Medical Research Council

Abstract: Antigen recognition by T cells is mediated by the multisubunit T cell antigen receptor (TCR) complex. Extensive research into signal transduction through the TCR has highlighted the complexity of this receptor. Subunits in the TCR complex involved in signal transduction contain 10 immuno-tyrosine based activation motifs (ITAMs), which are sequentially phosphorylated by the kinase Lck and dephosphorylated by phosphatases such as CD45. Phosphorylated ITAMs serve as docking sites for the cytosolic kinase ZAP-70, which has been shown to bind these ITAMs with differing affinities. Despite this extensive research, the purpose of multiple ITAMs, their sequential phosphorylation by Lck, and the differential affinity of ZAP-70 are unknown. The aim of this project is to use a detailed systems medicine approach to integrate these known TCR properties into a mathematical model. Predictions from the model will be tested experimentally and the model will subsequently be revised. A preliminary mathematical model that includes these TCR properties has suggested that the combination of these features (e.g. ITAM multiplicity, sequential phosphorylation, and differential ZAP-70 affinity) may be important for conferring sensitivity and discriminatory ability to the process of antigen recognition. In order to test these predictions, the TCR ITAMs will be systematically mutated and rearranged, and proximal and distal readouts of TCR signal transduction analysed. Based on these experiments, the mathematical model will be revised and calibrated so that it can be used to make further predictions. In addition, we will investigate the role of basic-residue-rich motifs that have been recently implicated in controlling ITAM phosphorylation in the underlying mechanism of sequential ITAM phosphorylation. Collectively, this combined mathematical and experimental work will enhance our understanding of the mechanisms and properties of T cell antigen recognition.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Targeting tissue macrophage generation as a mechanism to modulate the resolution of inflammation.
Funder: Medical Research Council

Abstract: We have identified transcription factors normally associated with development, cellular proliferation, differentiation and renewal that are selectively expressed in tissue resident macrophages (as opposed, for example, to monocyte-derived macrophages). We have shown that tissue resident macrophages are a self-renewing population that is independent of bone marrow-derived monocytic precursors. Since these transcription factors are anticipated or are known to be lethal when targeted to generate null alleles in mice, we will generate myeloid specific knockouts using the lysozyme M-Cre mice (Lyz2-Cre). These mice will be characterized for abnormalities in macrophage development and, assuming the mice are not fundamentally defective in tissue macrophages, in the response to inflammatory challenges. These challenges are designed to test their self-renewal and inflammatory responses at the same time. If feasible (depending on the severity of the impact of conditional-ablation on the macrophage lineage) the cells will also be tested ex vivo to establish if abrogation of these specific transcription factors alters phenotype in a way that fundamentally impacts on inflammatory responses, or is more restricted to roles in macrophage development and renewal. We believe that these studies will test the principle that we can specifically manipulate the existence and/or renewal of tissue resident macrophages, with only secondary impacts on the monocyte-derived lineage, and that this may provide a novel avenue to alter tissue homeostasis, host defense and the resolution of inflammation for therapeutic benefit.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Characterisation of the C-type lectin receptor CLECSF8 (CLEC4D)
Funder: Medical Research Council

Abstract: C-type lectin receptors (CLR) function in diverse ways and play essential roles in both immunity and homeostasis. We have identified a novel CLR which acts as an activation receptor and which is involved in anti-mycobacterial immunity. We will explore further the functions of this CLR so as to gain novel insights into the protective host mechanisms involved in immunity to Mycobacterium tuberculosis, which remains one the world's most deadliest bacterial infections. For these experiments, we will utilise a receptor-deficient mouse and explore the involvement of this CLR in cellular responses to M. tuberculosis in vitro, and in the induction of innate and adaptive immunity in vivo, using an aerosol model of infection. We will also investigate the functions of this CLR in adaptive immunity, using ovalbumin and ovalbumin-specific T-cell receptor transgenic mice. The ligand(s) and range of mycobacterial species recognised will be determined using cellular assays. We will translate our findings to the human system, by determining the function of the human CLR on primary and transfected cells, and the effect of various identified polymorphisms on receptor function. Ultimately we hope to explore the possibility of an association of these polymorphisms with alterations in disease susceptibility in human populations. We also have evidence that this CLR associates with a novel signalling adaptor molecule, possibly through interactions with its lectin domain. We propose several approaches to identify this adaptor, including methodology involving proteomic analysis and screening a cDNA expression library. Once the adaptor is identified, then subsequent analysis will involve understanding its role in CLR function and in anti-mycobacterial immunity. In sum, we have identified a novel receptor involved in anti-mycobacterial immunity, and the experiments described here will provide substantial and definitive insights into the role and functions of this CLR in both mice and humans.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Development of a high-content intracellular signalling pathway analysis methodology for drug repurposing
Funder: Medical Research Council

Abstract: We have developed a high-content protein analysis pipeline with high throughput potential, enabling components (50+) of multiple signaling pathways to be monitored simultaneously within large sets of biological samples. We will develop the methodology's potential as a drug repurposing tool for automated mid to high-throughput use. Repurposing of existing drugs for use in other diseases represents a highly cost effective route for pharmaceutical companies to gain new market opportunities for existing drugs and for re-examining failed compounds which showed promising characteristics. It also represents an important route to discovery of new therapeutics for a multitude of rare diseases, which are not economically attractive for major research and development by pharmaceutical companies. As a test case we will develop our platform to evaluate a compound set, consisting of marketed and off-patent drugs and known pharmacologically active compounds, for effects upon the defined intracellular signaling signatures which we have determined in the orphan autoinflammatory disease, TRAPS (TNF receptor-associated periodic syndrome). We have detailed knowledge of the signalling abnormalities caused by the presence of mutated TNF receptor 1 (TNFR1) in this rare syndrome. TRAPS associated mutations perturb the baseline balance of intracellular proinflammatory signals, resulting in hyper-responsiveness to encountered inflammatory triggers. No satisfactory small molecule drugs are available. Adequate treatment will therefore be best met by drugs that can restore signaling homeostasis. Screening for such drugs necessitates a multiplexed analysis of the perturbed pathways. Our platform will provide high-throughput, multiplexed reporting across a biomarker "landscape" of over 40 protein targets, suitable to such goals. It will provide a powerful, integrated system for re-examination and repurposing of drugs applicable to both orphan and common disease indications.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: NTD Highlight Notice: Discovering podoconiosis susceptibility genes: from molecules to disease control for a 'neglected' NTD
Funder: Medical Research Council

Abstract: Podoconiosis is a non-infectious geochemical disease that results in swelling of the lower legs. It is caused by long term exposure of bare feet to clay soil derived from volcanic rock. Podoconiosis is an important yet neglected public health problem particularly in tropical African countries. This proposal is underpinned by our programme of research on podoconiosis developed over the last 6 years, which has brought together a multidisciplinary team comprising expertise in clinical tropical diseases, epidemiology, public health, molecular genetics, bioinformatics and statistics. Having demonstrated that podoconiosis is heritable (63%) we have used a genetics approach towards identification of the molecular pathways involved in disease pathogenesis, which is a prerequisite for the development of better treatment for this stigmatising and debilitating disease. This proposal builds on the results of a genome wide association study (GWAS) that showed convincing association between variants in the class II HLA region and podoconiosis (P=1.42 x 10-9, OR=2.44). Suggestive association was identified in three non-HLA regions. In this proposal we intend to identify new population specific DNA variants both from the HLA region and across the exome to test for association in a larger cohort from the GWAS population and to replicate our findings in three new ethnically distinct populations from other regions in Ethiopia and Cameroon where podoconiosis is endemic. Experimental work will be undertaken at the Wellcome Trust Sanger Institute, making use state of the art core sequencing and genotyping facilities. In addition to the discovery of podoconiosis susceptibility genes, our work could shed light on mechanisms of disease pathogenesis for other HLA-associated diseases, and will generate large amounts of HLA data specific to African populations that will be valuable to the wider research community, for example those developing vaccines for important infectious diseases.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Anti-inflammatory cell contact-How do regulatory B cells sense and respond to apoptotic cells?
Funder: Medical Research Council

Abstract: We have previously reported that apoptotic cells protect in autoimmune models of arthritis by inducing regulatory B cells and our new data indicate that B cells secrete IL-10 in response to TLR9 signaling. Endogenous DNA complexes expressed on the apoptotic cell surface in nucleosomes are an important source of these signals. We wish to understand the B cell receptor specificity of these regulatory B cells both in murine and human B cells. We will examine B cells from healthy volunteers and in patients with rheumatoid arthritis and SLE. We have previously shown that B cells that respond to apoptotic cells make stable interactions with them prior to secreting IL-10 and FACs sorting will isolate these cells within the marginal zone and B1a B cell subsets. ELISPOT analysis will determine both the cytokine secretion profile and antigen specificity. Hybridomas will be generated following a standard fusion protocol and the B cell receptor specificity of the selected hybridoma antibodies will be determined by proteome array. A20 B cell lines will be generated using the heavy and light chain variable regions of selected hybridoma antibodies and tested for signaling focusing particularly on TLR7 and TLR9. The second aim of the proposal is to ask if accessory molecules such as C1q, sIgM and TIM4, which are associated with autoimmunity are crucial in mediating the regulatory response of B cells and this will be studied in cells deficient in these molecules.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The Role of Angiopoietins 1 and 2 in ANCA Associated Vasculitis
Funder: Medical Research Council

Abstract: Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is an autoimmune disease with a high morbidity and mortality. Key events in pathogenesis are thought to include neutrophil activation by ANCA leading to endothelial damage. Angiopoietins are growth factors essential in embryogenesis and maintenance of vascular integrity. More recently it has become increasingly evident that angiopoietins have a crucial role in inflammation. Previous data, which we have confirmed, has shown the angiopoeitin 2 is increased in the serum of patients with active ANCA vasculitis compared with controls. We hypothesise that an imbalance of these two angiopoeitins may potentiate disease in AAV. We will extend this study of angiopoeitins and related molecules in patient serum and renal biopsy samples. We will explore the effect of angiopoeitins on glomerular endothelial cells in vitro, and also in modulating the response of human neutrophils to ANCA in vitro. Our preliminary data suggests that angiopoeitin 2 augments the response to ANCA in TNF alpha primed neutrophils. We will also use a murine model of AAV and compare disease parameters in transgenic mice overexpressing angiopoeitin 1 or 2 with controls. This will be followed by experiments in which we explore the therapeutic potential of pharmacological blockade of angiopoeitin 2 in the in vivo model. We predict that these experiments will establish angiopoeitins as important factors in determining disease severity in ANCA vasculitis, and as potential therapeutic targets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The contribution of cutaneous lymphoid stress surveillance to atopy in human health and disease
Funder: Medical Research Council

Abstract: Background: The NKG2D-mediated lymphoid stress surveillance response (LSSR) is implicated in the innate immune response to epithelial damage. Murine data has shown that activation of this pathway in the skin can influence the response to cutaneous antigens inducing a strong TH2 atopic response. Aims: To investigate the contribution of cutaneous lymphoid stress surveillance to atopy in human health and disease Objectives: 1. To understand the mechanism by which certain cutaneous sensitisers induce NKG2D ligand (NKG2DL) expression by keratinocytes. 2. To interrogate the pathway that may promote this response through the isolation, characterisation and functional investigation of human skin-resident NKG2D+ cells. Methodology: Flow cytometry and qPCR will be used to establish if NKG2DL expression induced by cutaneous sensitisers is regulated at a pre or post-translation level (HaCaT and primary human keratinocytes). If post-transcriptional mechanisms are indicated microRNAs that bind NKG2DL mRNA will be investigated by qPCR. Cell signalling pathways that may alter NKG2DL expression will be also be investigated (such as DNA damage-response pathway). Skin-resident lymphocytes will be isolated from whole skin by direct out-growth from epidermal sheets or out-growth from whole skin onto bioengineered matrix. NKG2D+ cells (FACS-isolated) will be characterised by flow cytometry and functionally interrogated in vitro by co-culture assay. To investigate the role of the tissue environment lymphocyte responses will be correlated with filaggrin genotype. Scientific and Medical Opportunities: This project will provide important new information about the human skin-resident lymphocyte compartment and NKG2D-mediated LSSR to environmental allergens. It will help establish if this pathway can influence the TH2 atopic response in man. This has wider implications for our understanding of the innate human response to epithelial damage and will lead to novel clinical studies.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM