NIHRDataInsights/HRCSData huggingface dataset:
Title: The Fundamentals of Phagocytosis: Integrating Theoretical Models and Experiments
Funder: Medical Research Council

Abstract: Phagocytosis is still poorly understood, despite being first described over 100 years ago. For example, the membrane forces and signalling involved, the precise role of the actin cytoskeleton, and the effect of target shape and size are almost completely unknown. This is surprising since these issues are crucial in multiple issues of human health, including the design of microparticle drug delivery systems and various medical conditions such as Wiskott-Aldrich syndrome. Here I plan to use a multidisciplinary, integrative approach that combines theoretical studies (mathematical modelling, physical processes and computing) with dual-micropipette experiments that allow the exact shape of the phagocytic cup to be studied with >1Hz time resolution. In particular, I will, for the first time as far as I am aware, use membrane models (with the membrane represented by a series of points connected by springs) that include signalling (e.g. Meinhardt-like activator-inhibitor pairs) and actin (via a realistic branched network structure). These models will be informed by and compared to real time-lapse movies of phagocytosis, which will be obtained from a dual-micropipette setup. This setup allows precise control over both the immune cell (e.g. mice RAW 264.7 macrophages or neutrophils extracted from whole blood) and the target (e.g. IgG-coated polystyrene beads of various shapes and sizes). This dual theoretical-experimental approach will allow various questions to be addressed including (i) the role of target shape and size, (ii) the effect of target orientation, (iii) the role of actin and F-actin branching, (iv) the details of the multistage nature of the engulfment rate, (v) the role of different types of signalling molecule, and (vi) the similarities and differences between phagocytosis and EPEC pedestal formation. Perhaps the most exciting outcome will be direct predictions for the optimal shape and size for microparticle drug systems.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Defining and exploiting the role of PML protein in innate immune responses to pathogens
Funder: Medical Research Council

Abstract: This project will define and elucidate the role of PML-II protein in the transcription of genes induced during interferon (IFN) and inflammatory responses, determine whether this role represents a source of natural variation in these responses and whether it can be targeted to tune their potency. We will use the newly emergent CRISPR/Cas9 approach to generate isogenic cells that are either capable or incapable of expressing PML-II. These cells will be compared, using state-of-the-art RNAseq to analyse mRNA populations following treatment with IFN inducers or IFN itself. Similarly, we will use ChIP-seq to analyse the binding of key transcription factors (TFs) to chromatin that follows the application of these inducers, with or without PML-II. Thirdly, we will use stable isotope labelling (SILAC)-based quantitative proteomics to determine which proteins interact with the unique C-terminal domain of PML-II, which we have shown previously to be necessary for its function in transcription and for its association with specific TFs and co-activators. We will then correlate these data sets using powerful systems analysis tools to determine which TFs mediate PML-II-dependent responses. Natural variants of PML-II will be cloned by standard techniques and their ability to support robust innate immune response gene activation assessed by RT-qPCR analysis of mRNA from transfected cell cultures subjected to innate immune stimulation, an established assay of PML-II function. Variation in PML isoform expression and assembly in the cell will also be induced by short and long-term mild heat stress and the effects on the ability of cells to support virus infection and to mount innate immune responses assessed using immunofluorescence, RT-qPCR, western blotting and cytokine ELISA. Finally, short segments of PML-II will be generated by cDNA cloning and expression, and tested as potential inhibitors of the innate response, measured at the mRNA level using RT-qPCR.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Structural and Functional Roles of Transmembrane Domains in B-cell Receptor Signalling
Funder: Medical Research Council

Abstract: We undertake here to investigate the transmembrane domain (TMD) interactions involved in assembly of the B cell receptor complex, since these regions have been discussed as sites of functionally essential (but poorly understood) protein-protein and protein-lipid interactions for the past 25 years. We hypothesise that the TMDs of the three proteins in the BCR complex are mediators of strong and specific (yet uncharacterised) interactions. The aim of the work described in our proposal is to provide the first comprehensive map of TMD-TMD interactions possible in the BCR complex in natural membranes as well as synthetic membranes with varying lipid composition. The three main objectives of the work are (i) characterisation of the strength (stability) and sequence dependence of TMD interactions within (e.g IgM homodimers, CD79a-b heterodimer) and between (e.g. CD79-IgM) components of the BCR complex in a natural membrane using the TOXCAT and GALLEX assays and scanning mutagenesis, (ii) utilisation of a panel of biophysical methods (including circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation and solution state NMR) to characterise the structure, stability, stoichiometry and atomic details of TMD interactions in multiple (synthetic) lipid environments, and (iii) use of this new information to design and test ability of molecules to disrupt TMD interactions and modify BCR signalling and function in situ in the lab of Prof. James Drake (Albany Medical College, New York). Molecules will be applied to B cell lines of murine splenic B cells to determine their impact on signalling and endocytosis. Commercial viability of downstream applications of our work (e.g. molecules for modulation of BCR function) will be assessed with support from Warwick Ventures, our technology transfer office.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: IMPC: Use of IMPC knockout mice and novel transgenic murine models to identify molecular pathways controlling thymic regulatory T cell development
Funder: Medical Research Council

Abstract: The immune system provides an essential protective role against bacterial and viral infection, and additionally plays a key surveillance role in guarding against tumour formation. A critical immune cell type involved in this process is the T cell. However, T cells not only play a critical role in protective immunity, but if incorrectly regulated can potentially attack the body's own tissues and cause autoimmune disease. The thymus is an organ located within the upper chest that provides the main anatomical site for T-cell development. During T cell development, T-cells are screened for their potential to mount an immune response against our own tissues. Those T-cells bearing a high likelihood of attacking the body's organs are forced to undergo cell death. However, this process is not 100% efficient, meaning that T-cells with the potential to cause autoimmune disease are able to escape deletion within the thymus. In order to keep autoreactive T-cells in check, the thymus additionally supports the development of a highly specialised type of T-cell, termed regulatory T-cells (also called Treg). Regulatory T-cells possess a unique capacity to supress the activity of other T-cells, including autoreactive T-cells. In addition, regulatory T-cells also curtail protective T-cell responses following the clearance of infections, which if left unchecked may also cause 'friendly-fire' damage to bystander tissues. Regulatory T-cells generated within the thymus therefore play a critical role in controlling the activity of the immune system and therefore impact autoimmune disease. However, the precise mechanisms that control regulatory T-cell development and function remain incompletely understood. This project aims to use novel experimental approaches to identify new molecules that control the development and function of regulatory T cells generated within the thymus. We aim to use innovative methods that will allow us to accurately identify regulatory T cells undergoing development within the thymus and quantitate their output from the thymus into the peripheral circulation when the expression of individual defined genes are defective. The identification of new molecules controlling regulatory T cells development and function may ultimately inform future approaches to therapeutically manipulate regulatory T cell and therefore modulate immune system activity in health and disease.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Neutrophil Vesicles as a novel autologous regenerative therapeutic for joint disease.
Funder: Medical Research Council

Abstract: We are seeking support to define the molecular and cellular pathways activated in vivo by neutrophil-derived extracellular vesicles (PMN EVs) to enable cartilage repair during joint inflammatory arthritis. This is the main thrust of this application. Supported by our recent publication, and a series of unpublished data, we propose a 2-year integrated research programme to define - 1. Efficacy, and mechanisms behind it, following intra-articular injection of PMN EVs in inflammatory arthritis. 2. Minimally required criteria in specific assays to establish inter-preparation variability/quality controls for efficacy and begin to pave the way to further development for this therapeutic approach. 3. The biomolecular mechanisms triggered by PMN EVs prepared by patient suffering from rheumatoid arthritis. As such we will use one model of joint disease (for mechanistic purpose, to identify new markers of efficacy, complementing assessment of cartilage status with monitoring of inflammation and structural damage), one model of ectopic cartilage implantation (to assess translational potential for PMN EVs in human tissues) and a series of in vitro assays to establish the required degree of in vitro efficacy to predict efficacy in vivo. These in vitro assays will be applied also to define the bio-actions of PMN EVs produced from rheumatoid arthritis patients. This project will deliver the biological information and quality controls required for further development of PMN EVs as a novel autologous regenerative therapy.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES;6.2 CELLULAR AND GENE THERAPIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Transitional B cell development in health and systemic lupus erythematosus
Funder: Medical Research Council

Abstract: 1. Perform a deep transcriptomic analysis of transitional and mature naive B cell populations using RNA-sequencing. Cells from healthy donors will be isolated and sequenced to identify expression of a large number of potential homing receptors and how these change through B cell development. 2. Validation of RNA-sequencing by quantitative polymerase chain reaction (qPCR) and flow cytometry on further healthy controls. This will allow quantification of specific lineage associated markers of migration in health, that will allow us to generate a panel of markers to investigate TS cell migratory abnormalities in SLE. 3. Perform deep phenotypic analysis of transitional B cells in patients with different clinical subtypes of SLE. We hypothesise that different homing receptor phenotypes in SLE may concentrate transitional B cells in abnormal lymphoid compartments, and may predisposed individuals to certain patterns of organ involvement. To investigate this further Time of flight mass cytometry (CyTOF) with a panel of 40 lineage associated markers of maturation and migration will be used to interrogate immature B cell subsets. Patients will be divided into three cohorts according to organ involvement, these being predominant nephritis, arthritis and cutaneous disease. Where differences are seen, these will be confirmed by flow cytometry and quantitative PCR on further SLE patients.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The oRAcle Study - Predicting the risk of infection in RA
Funder: Medical Research Council

Abstract: Background: Patients with rheumatoid arthritis (RA) experience frequent and severe infections. The current dogma is that infection is a result of the pharmacological agents used to treat the disease. Evidence for this is not robust. It is recognised that the inflammatory state in RA is immunosuppressive. This is supported by patients with active disease having more infections. We hypothesise that the active disease state in RA is the dominant risk factor for infection. Aim: To investigate the extent to which disease-related immunological signatures are predictors of infection. Objectives 1.To characterise infection in early RA 2.To determine the cellular and molecular signatures that are predictors of infection 3.Develop a therapy decision tool that stratifies for infection risk Methods: A dataset derived from an established early RA cohort will be used (TACERA). This contains extensive clinical, cellular and molecular data on 270 patients with RA and a comprehensive prospective capture of all infectious events from diagnosis to 18 month follow-up. A Delphi exercise will be conducted to support grading of infectious events. A case control approach will be used to identify clinical, cellular and molecular signatures associated with infection. Validation analysis will include an 'extreme infection' phenotype as well as a survival modelling of 'time to event' approach. Cellular and molecular signals associated with infection will be analysed at 6 months after the initiation of DMARDs to assess the impact of drug therapy on infection. The large SERA cohort (n=1200) will be used to validate the results of this study. Scientific opportunities: A clinically valuable risk prediction tool will be developed using a biomarker sets based on the cellular and molecular signatures that best predict infection. This will contribute to a web based 'infection risk score' to identify 'high-risk' patients and inform decisions regarding RA drug therapy and infection.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of Mediator in T-bet-dependent gene activation and its dysregulation in mucosal inflammatory disease.
Funder: Medical Research Council

Abstract: 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.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: Myeloperoxidase inhibition as a potential therapy in anti-neutrophil cytoplasmic antibody vasculitis
Funder: Medical Research Council

Abstract: ANCA vasculitis a severe systemic disease which affects joints, lungs, kidneys, skin and other tissues. It occurs most often in older adults. Autoantibodies against the neutrophil and monocyte antigens myeloperoxidase (MPO) and proteinase 3 are found in patients and in vitro and in vivo data suggest that these play a key role in pathogenesis. Our hypothesis is that inhibition of MPO enzyme activity will be an effective therapy for ANCA vasculitis via effects on both neutrophils and monocytes. We plan to test this through a combination of experiments using human monocytes in vitro and murine in vivo models. The in vivo model will include the murine model of anti-MPO vasculitis in which crescentic glomerulonephritis is induced by injection of anti-MPO antibodies raised in MPO deficient mice. In addition to establishing the potential of this approach for therapy, we will explore some novel mechanisms by which MPO inhibition may affect monocyte cell function.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Molecular regulation of necroptosis in inflammatory diseases.
Funder: Medical Research Council

Abstract: This project will assess the role of programmed necrosis, also termed necroptosis, in inflammatory diseases and seek to identify necroptosis inducers in pathophysiological conditions. Necroptosis has previously been shown to trigger inflammation in multiple animal disease models. To date, the role of necroptosis has been poorly investigated in human diseases. Recently, I have identified alarmin-triggered necroptosis as a central mechanism in the pathophysiology of skin inflammation in Toxic Epidermal Necrolysis (TEN). This Study will investigate the mechanisms of necroptosis activation by alarmins in epidermal keratinocytes. Experiments will, in a first place, use in vitro cell death assays (Wst1, CytoTox Glo, caspase assays), immunofluorescence and live imaging to monitor cell death. Then, we will seek to identify the components of the alarmin-induced necrosome, using proteomics and phosphoproteomics analysis. The role of alarmins will then be assessed in in vivo models of necroptosis-induced skin inflammation, by means of genetic crosses or use of blocking antibodies. The improvement of skin inflammation will be evaluated by macroscopic observation and histological analysis. Finally, we will extend the study of the role of necroptosis and alarmins in inflammatory diseases to a broader spectrum of skin and joint inflammatory diseases, thanks to a collaboration with clinicians at University Hospital of Wales, as well as models of pancreatitis and Alzheimer disease, using the biomarkers that I have previously developed. The results of this study are expected to demonstrate the role of necroptosis in inflammatory diseases and decipher its molecular regulation by alarmins in vitro and in vivo, opening the way to new therapeutic approaches for inflammation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Innate immune regulation of infection tolerance
Funder: Medical Research Council

Abstract: Hosts survive infection by engaging mechanisms that combine resistance and tolerance. Resistance mechanisms include responses to infection that protect the host by killing pathogens or restricting their growth; this is the immune response as we normally envision it. In contrast, tolerance is defined as the ability to maintain health during infection; experimentally, a more tolerant host is one that remains healthy longer at a given pathogen load. Recent years have seen increasing interest in tolerance, driven in part by the idea of developing tolerance-based infection therapies. We show here that Drosophila melanogaster imd mutants exhibit a strong tolerance defect when infected with two different bacterial pathogens. imd encodes the Drosophila homolog of the TNF pathway component RIPK1 and plays a well-established role in infection resistance; the fact that we observe a tolerance defect in these infections implies that infection-induced imd signalling triggers infection tolerance as well as resistance. The experiments we propose will identify the mechanisms underlying imd-driven infection tolerance. We propose three aims. First, we will identify the physiological parameters associated with the tolerance difference between imd mutants and wild-type flies. Second, we will determine in which tissues imd is required to promote infection tolerance and which imd signalling mechanisms are relevant to tolerance. Third, we will identify the genetic mechanisms acting downstream of imd by functional screening of imd target genes; we will then identify the specific functional connections between imd targets, the physiological effects identified in the first aim, and realised infection tolerance. By identifying the genetic and physiological mechanisms behind imd-driven infection tolerance, we will shed light on in vivo mechanisms protecting animals from damage due to infection.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM