NIHRDataInsights/HRCSData huggingface dataset:
Title: Integrating Capture-HiC with omic time course data to uncover the regulatory interactions modulated by genetic variation in disease
Funder: Medical Research Council

Abstract: It has become clear that T-cells are of fundamental importance in Rheumatoid arthritis (RA). In this project we will use time course data from stimulated primary human T-cells to uncover the regulatory function of RA-associated enhancers controlling target gene expression. Capture-HiC data enriched for promoters and disease-associated enhancers will be used to identify the physical interactions between enhancers and promoters and how these change through time. Nuclear RNA-Seq will be used to quantify nascent transcription. ATAC-Seq will be used to profile open chromatin and identify likely transcription factor binding (TF) events. We will collect data from these three assays from the same primary T-cell population over a 24 hour time period after stimulation. We will then use these data to develop a regulatory network model, using non-parametric Gaussian process methods to infer TF activities and mathematical models to describe the effect of TF-binding on promoter activity and nascent RNA production rates. We will score alternative regulatory network models through Bayesian model comparison in order to identify the most well-supported models of regulation for each target gene. Computational methods will be scaled up from previous smaller scale applications to deal with more complex enhancer-driven regulation involving large numbers of regulatory TFs. Methods will be published as open source computational tools for the community. Finally, we will validate our predictions through follow-up experiments on human data by comparing subjects with differences in SNPs implicated through our model-based analysis. We will use targeted ChIP-PCR to look at the effect of these SNPs on epigenetic marks and TF binding and targeted 3C to confirm enhancer-promoter interactions and look at whether these are altered according to genetic differences.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Understanding the regulation of thymus function to control self-tolerant T-cell production
Funder: Medical Research Council

Abstract: Building on our long term research interests, we propose to investigate the cellular and molecular regulators of self-tolerant T-cell production in the thymus. Having established the functional dichotomy of the cortical and medullary epithelial lineages, and shown that they derive from a common precursor pool, we will seek to define the precursor pool in both mouse and man, the molecular mechanisms that determine their lineage specificity, and study how these microenvironments select the developing T-cell receptor repertoire. We will build on our recent discovery of the impact of CD1d-restricted iNKT-cells on the thymus medulla, to understand how these cells and their products influence thymus function and tolerance induction. Finally, we will extend our knowledge of the mouse thymus to study and manipulate human thymic epithelial cells and their progenitors, and define novel approaches to measure thymus function in man. Such studies will facilitate future therapeutic manipulation of human thymus function for effective T-cell production in man. Our specific goals are as follows: 1. To use newly generated RANK-Venus reporter mice to isolate and define the TEC progenitor populations that give rise to cortical and medullary TEC compartments across the life-course in the mouse. 2. To understand the impact of immunoregulatory CD1d-restricted iNKT-cells and their products on the formation of tolerogenic thymic microenvironments. 3. To determine whether induction of the Unfolded Protein Response by mTEC is linked to their ability to induce T-cell tolerance. 4. To extend our murine studies towards the identification of human thymic epithelial progenitors and establish approaches that enable the analysis and manipulation of thymus function in man.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Investigation of the early growth response gene (Egr) 2 and 3 mediated regulatory programme in T cells
Funder: Medical Research Council

Abstract: Clonal expansion and differentiation of effector cells are hallmarks of T cell responses against virus infection. Dysregulated expansion and/or differentiation can result in chronic infection, autoimmunity and severe immunopathology. We have discovered that the transcription factors Egr2 and 3 have an overlapping function in promoting clonal expansion, but restraining differentiation of effector T cells, in response to virus infection and also suppressing inflammatory responses of self-reactive T cells under homeostatic conditions. Defects in both Egr2 and 3, but not single Egr2 or 3 deficiency, in T cells results in defective anti-viral responses with high inflammation which resembles the altered T cell function in chronic infectious. Intriguingly, T cells in autoimmune disease also have a poorly proliferative, but highly inflammatory, phenotype. We found that Egr2 and 3 are only expressed in a subpopulation of CD44high T cells. Using our models of CD2-Egr2Ko, CD2-Egr2/3Ko, CD2-Egr2 transgenic and GFP-Egr2 knockin mice, as well as Egr3Ko, we will dissect the mechanisms of Egr2 and 3 function in T cells and investigate; the function of Egr2 and/or 3 expressing T cells in homeostatic and anti-viral responses, the mechanisms of Egr2 and 3 function in effector T cells for reciprocal regulation of proliferation and differentiation during anti-viral responses, and the molecular programmes regulating Egr2 and 3 expression in effector T cells under homeostatic conditions and during anti-viral responses. The results will impact not only on our understanding of T cell responses, but may also provide mechanisms underlying dysregulated immunoresponses in chronic infection and autoimmune diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: Investigating the pathogenic role of T follicular helper cells and their therapeutic targeting in primary Sjogren's syndrome
Funder: Medical Research Council

Abstract: In this proposal we will test the hypothesis that T follicular helper (Tfh) cells fuel autoreactive B cell activation and autoimmunity in Sjogren's syndrome (SS), in particular in patients with germinal centres (GC) in the salivary glands (SG). Moreover, we aim to pursue with our industrial partner a translational program to develop Tfh blocking agents for clinical use in SS and to understand how Tfh-based patient stratification may inform randomised clinical trial (RCT) design. We will proceed following 3 progressive steps: 1) To investigate the presence of Tfh and their products in the circulation and SG of SS and characterise their functional role in promoting autoreactive B cell activation. We will access the deeply phenotyped EMR SS Biobank with over 150 SG biopsies with matching histology, RNA and peripheral blood. To identify Tfh-related signatures and stratify samples according to the degree of lymphoid organization, we will use: i) multicolour confocal microscopy ii) Fluidigm microfluidic chips iii) multicolour FACS analysis iv) in vitro organ cultures 2) To test in vivo the efficacy of pharmacological blockade of Tfh in modulating sialoadenitis, autoimmunity and exocrine dysfunction in murine models of SS. We will block key factors involved in Tfh function, such as the ICOS/ICOSL and the IL-21/IL21R pathways, in order to ameliorate disease in 2 animal models of SS: i) spontaneous NOD.H2h4 mice ii) novel human SS/SCID xeno-grafts 3) To define the contribution of Tfh to SG pathobiology and disease outcome through the analysis of biopsies pre- and post-treatment in order to inform Tfh-based patient stratification. We will use matching SG biopsies and peripheral blood collected as part of the UK multicentre RCT of Rituximab in SS. Using the techniques summarised in Aim 1, we will investigate whether: i) Tfh signatures are influenced by B cell depletion ii) persistency of Tfh allows the escape of autoreactive B cells in the SG


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Contribution of Neuro-inflammation to Cerebral Ischaemia
Funder: Medical Research Council

Abstract: Inflammation that occurs in response to tissue injury in the absence of pathogen is considered sterile, and can contribute to damage. The primary inflammatory cytokines associated with sterile inflammatory responses are members of the interleukin-1 (IL-1) family, IL-1a and IL-1b. IL-1 release from activated immune cells is regulated by a protein complex called the inflammasome. IL-1 is expressed in response to damage associated molecular patterns (DAMPs) that stimulate pattern recognition receptors (PRRs) on cells of the innate immune system. Once expressed, pro-IL-1 remains intracellular until an additional DAMP stimulation activates inflammasomes. Inflammasomes activate caspase-1 which in turn processes pro-IL-1 allowing release of the mature cytokine. We have discovered that brain inflammation after cerebral ischaemia is dependent upon the AIM2 and NLRC4 inflammasomes and also requires the inflammasome component ASC. However, we do not understand whether these inflammasomes are activated in the brain, whether they drive systemic inflammation, or indeed whether they contribute directly to cell death. We aim to identify the cell type and location driving the respective inflammasome-dependent responses, and to dissect the inflammasome-dependent neurotoxic and inflammatory responses activated by CI. We will use a number of complimentary approaches to achieve the aims of this proposal. The middle cerebral artery occlusion (MCAo) model of stroke is a robust and clinically relevant model which we will use in mice in which components of different inflammasomes (e.g. AIM2, NLRP3, NLRC4, ASC) have been knocked out. Inflammatory, injury, and behavioural endpoints will provide a comprehensive view of the role of these proteins in the inflammatory response. We will also use functionalised carbon nanotubes (f-CNTs) to deliver RNAi to specific brain areas to uncouple the inflammatory response from cell death, and so fully interrogate mechanisms of inflammasome function.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The Role of 18kDa Translocator Protein (TSPO) in cellular bioenergetics and microglial activation
Funder: Medical Research Council

Abstract: Aims: Understand the mechanism by which TSPO ligands modulate microglial phenotype, and investigate effect of rs6971 polymorphism Objectives 1. Establish IPSC derived microglia in my lab and characterise the cellular bioenergetic profile of these and primary human microglia 2. Characterise protein-protein interactions of microglial TSPO 3. Test the hypothesis that modulation of microglial immunophenotype by TSPO ligands is mediated by an effect on cellular bioenergetics. Methods Aims 1 and 3: The respiratory chain complexes will be analyzed using Western blot and in vitro activity assays of the native and denatured whole mitochondrial protein samples. The complex enzymatic activity will be assessed in vitro. The activity of Complex-V will be assessed using the NADH or the luciferase assays. To determine the coupling of mitochondrial oxidation to phosphorylation, the leak currents will be measured by patch clamp electrophysiology on the mitochondrial inner membrane vesicles. On-organelle patch clamp recordings at holding voltages between -100 mV and +100 mV will be obtained. ROS will be measured with CellROX Green and Mitosox Aim 2: Myc and FLAG-tagged constructs for the wildtype and rs6971 mutant TSPO will be overexpressed in control and activated IPSC-derived microglia. Mitochondria will be isolated. TSPO will be immunoprecipitated and candidate proteins (already identified by the phylogenetic profiling) will be assessed by using LC-MS/MS Scientific/Medical value of results A treatment capable of protecting against neurodegeneration would have clear medical benefit This work will foster new research, not only in neurodegenerative diseases, but also in aging, stroke, encephalitides and certain psychiatric diseases (eg schizophrenia), all characterised by increased TSPO expression


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Functional investigations of the influenza virus proteome.
Funder: Medical Research Council

Abstract: This research aims to analyse influenza virus infections at a molecular level by combining proteomic studies with molecular biology and classical virology. It has two main objectives: to relate the protein composition of influenza virions to their ability to infect cells, and to relate changes in viral and host protein phosphorylation during the course of an infection to the regulation of viral replication. The methodology integrates descriptive mass spectrometry (MS) with hypothesis testing in molecular virology. MS will be used (i) for absolute quantitation of proteins in complex mixtures (virions and whole cell lysates), (ii) to map protein phosphorylation and (iii) for relative quantitation of proteins from different time points during infection. I have experience of applying the first two approaches and I have already used them to generate large datasets relevant to the proposal. I also have preliminary data demonstrating the technical feasibility of the third approach. Most importantly, I have experience of combining these large MS datasets with evolutionary, structural and functional data to produce testable hypotheses about virus biology, and I will recruit bioinformatic support to infer hypotheses about more complex cellular pathways. I will then relate protein composition to infectivity by virus titration, protein overexpression and knockdown, studies of interferon induction and (in collaboration) electron cryotomography. For signalling pathways which show changes in phosphorylation during infection, I will knock down or inhibit key proteins in these pathways to test whether they regulate viral growth or the localisation and activity of viral proteins. I will pay particular attention to findings that will inform the development of antiviral kinase inhibitors. Together, these approaches will provide an opportunity to deepen our scientific understanding of influenza viruses and to develop strategies for their control.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Re-engineering of fungal sulphur metabolism to limit mould viability and virulence.
Funder: Medical Research Council

Abstract: I will seek a mechanistic understanding of A. fumigatus in vivo S-requirements using state-of-the-art NanoString gene expression technology of fungal RNA isolated from infected mice. I have demonstrated that methionine (met) synthase is essential in A. fumigatus. I will next attempt to construct a met auxotroph in which met synthase activity is maintained (metG cysD double mutant), to ascertain whether met is a vital in vivo S-source and met biosynthesis per se is essential for A. fumigatus. I will seek specific inhibitors of fungal cobalamin-independent met synthases, and assess their relevance as antifungal therapeutics. I will test polyglutamated folate analogues and I will also screen a MRCT library of 9000 compounds held at the Manchester Fungal Infection Group (MFIG), using an in vitro, spectrophotometric method. Drug candidates will then be tested to confirm their impact upon fungal growth using a luciferase expressing A. fumigatus strain. Drugs having an inhibitory effect will be further tested in animals to evaluate their suitability for treatment. I will investigate the relevance of H2S, a gas produced in mammalian tissues, for A. fumigatus growth and virulence. I will test virulence of a cysD cysB double mutant, which should not incorporate H2S, to determine if H2S is exploited as S-source. Since H2S has been described to target cysteines, a protein modification termed sulfhydration, I will also ascertain whether this occurs in A. fumigatus. I will adapt two previously described methods to compare protein extracts from wt and a mecA mecB double mutant (which I hypothesise would be a sulfhydration-defective strain). Furthermore, I will utilize a mouse CSE-/- line, defective in H2S production in peripheral tissues, to confirm the relevance of this gas in vivo. Finally, I will seek correlations between fungal S- assimilation and/or human S-status, and the occurrence of disease using the database of genome sequences for aspergilloses curated at the MFIG.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Testing the Mechanism of T lymphocyte selection in the thymus mediated by the zfp36 family of RNA binding proteins
Funder: Medical Research Council

Abstract: A significant body of unpublished data form the applicant's laboratory indicate that RNA binding proteins (RBP) of the zfp36 family play a role in thymic development and selection. We want to find the mechanism through which these RBP function. We will use well-characterised TCR transgenes which will allow lineage choice to be monitored. Importantly, the TCRs chosen will enable experiments to be performed using peptide MHC stimulation delivered by APC or tetramers and for the OT1 TCR there are a set of peptides of varying affinity which mediate positive and negative selection of thymocytes. These peptide MHCs will also allow signal transduction to be tested in a variety of assays such as imaging calcium flux or the phosphorylation of specific pathway components. The role of cell death will be examined by using a bim knockout system that will rescue cells dying from neglect and negative selection. Alternative/additional hypothesis including altered cytokine signalling can be tested using antibody based approaches for probing signal transduction (e.g. intracellular flow). The use of a GFP reporter transgene (GFP-Nur77) which acts as a reporter of TCR signal strength will allow us to examine how the signal strength of this receptor changes during development and in the absence of the RBP. This class of RBP is able to bind to the ARE which is found in the 3'UTR of10% of mRNAs. Bioinformatics predictions based on sequence features alone have failed to identify the consequential targets of these RBP making it essential to define direct targets through empirical measurements. To do this we will use the iCLIP method which is well-established in the Turner lab. We will then discover how direct and indirect targets are changed in abundance using RNAseq data. Overall, this project will allow the integration of an important new layer of control of gene expression into our understanding of the molecular regulation of thymocyte development.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Control of macrophage multinucleation in health and disease
Funder: Medical Research Council

Abstract: We hypothesise that key genetic determinants of macrophage multinucleation orchestrate fundamental signalling pathways that regulate bone resorption and are perturbed in granulomatous disease. We will use genome-wide expression and systems-genetics, rapid-throughput phenotyping, and disease provocation models in order to: 1. Undertake an RNAi screen and RNAseq in primary macrophages to identify key genetic determinants and pathways regulating macrophage multinucleation 2. Identify 10 genes with the largest inhibitory effect on macrophage multinucleation, and establish their physiological importance using skeletal samples from corresponding knockout models obtained from the IKMC 3. Select two genes on the basis of effect size, tractability, function and translation to human disease and investigate the molecular mechanisms of macrophage multinucleation 4. Generate monocyte-macrophage specific knockout lines for the two selected genes, perform detailed skeletal phenotyping and use provocation models for osteoporosis, inflammatory arthritis and glomerulonephritis We will use siRNA knockdown for each of 134 genes that comprise a network controlling macrophage multinucleation. We will identify 10 genes with the largest effect and perform RNAseq and systems-level bioinformatics to determine the key signalling pathways essential for multinucleation. Skeletal consequences of global deletion of these 10 genes will be determined by X-ray microradiography, micro-CT and biomechanical testing. Analysis of developmental and adult phenotypes will be performed in two macrophage specific knockout lines using additional methods including electron microscopy, histomorphometry and primary cultured osteoblasts and osteoclasts. Ovariectomy, collagen-induced arthritis and macrophage-dependent crescentic glomerulonephritis disease models will be used to determine the effect of gene deletion on susceptibility to osteoporosis and chronic inflammatory joint and kidney disease.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: Treatment According to Response in Giant cEll arTeritis (TARGET)
Funder: Medical Research Council

Abstract: Giant cell arteritis (GCA) is the commonest primary systemic vasculitis. Ischaemic complications, including visual loss, occur in 19%. Large-vessel vasculitis leads to stenoses and aneurysms. GCA is difficult to diagnose; to prevent blindness, suspected GCA is treated empirically with high-dose glucocorticoids. However blood and imaging tests can normalize within days of therapy, whereas temporal artery biopsy is insensitive. 86% of patients have adverse effects with glucocorticoids. Glucocorticoids can trigger psychosis or arrhythmia within days, prompting further admissions and treatments. Many other adverse events accrue over time and 50% still need glucocorticoids at 2-3 years. There is little trial data to guide adjunctive immunosuppression, and these data are equivocal due to patient heterogeneity and imprecision of outcomes. We will develop methods for phenotyping patients to accurately identify the severity of organ involvement, the dominant immunological pathways, and risk factors for glucocorticoid toxicity. Integrating this information, leveraging existing and current datasets, will allow modelling of the most appropriate treatment pathways and rational patient stratification. We will also use outcome measures to identify good prognosis, non-relapsing groups in order to minimize therapy and reduce toxicity in these patients. Refinement of the molecular and immunological phenotype will also allow rational patient selection for early-phase clinical trials of selected biological therapies supporting drug repurposing, subject to target validation. In parallel, we will generate contemporary epidemiological data to support clinical adoption of emerging biomarkers or therapeutic strategies through incorporation of health economic and cost-effectiveness analyses and engagement with NICE. Future work will include the development of virtual training material and expert centres, supported by NHS England, through specialist commissioning centres.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM