NIHRDataInsights/HRCSData huggingface dataset:
Title: Regulation of inflammation by homeostatic cholesterol transport pathways.
Funder: Medical Research Council

Abstract: Over the past several years, emerging evidence has suggested the tight regulation of immune cell function and inflammatory pathways by cellular lipid metabolism. Inflammasome, a multi-protein complex, is recognised as having pivotal roles in inflammatory and autoimmune diseases. Assembly of the NLRP3 inflammasome leads to caspase-1 processing and IL-1b secretion, which initiate an inflammatory reaction with implications in several conditions including cardiovascular disease as demonstrated by the recent CANTOS trial. The cellular metabolic pathways that regulate inflammasome are not well defined. Our exciting new data suggest that cellular cholesterol trafficking licenses NLRP3 activation. Cholesterol requirement of cells is achieved through biosynthesis and uptake programs. In an intricate pathway involving lysosomal cholesterol transporter NPC1, the sterol gets unequally distributed across intracellular compartments. Specifically, we demonstrated that blockade of NPC1-dependent cholesterol efflux through the late-endosome/lysosome pathway dampens NLRP3 activation (Journal of Cell Biology, in revision). We now propose to decipher detailed cholesterol transport pathways that are central to inflammasome activation and gain new understanding into the modulation of NLRP3 activation by cholesterol metabolism. By employing biochemical approaches, we will characterize the sub-cellular organelle to which cholesterol must localize for optimal inflammasome activation. Moreover, we will use the experimental model of monosodium urate (MSU) crystal-induced peritonitis to shed light on the physiological role of the identified cholesterol trafficking pathways in NLRP3 activation. Together, our studies will reveal novel metabolic regulators of the NLRP3 inflammasome and identify upstream targets to manage overt inflammation in clinically important diseases.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Therapeutic targeting of fibroblast subsets in inflammatory arthritis
Funder: Medical Research Council

Abstract: In this programme we will use functional single cell based approaches combined with fate mapping techniques in mice to test the hypothesis that distinct synovial fibroblast subsets, residing either in the lining or sub-lining layer, differentially mediate synovial inflammation and tissue damage; which we propose can then be selectively therapeutically targeted. To do this we will initially explore how the fibroblast subsets that we have identified change during the development of arthritis and during the switch from resolving to persistent disease in order to determine the functional consequences of these changes in relation to inflammation, damage and repair. Next we will determine the developmental origins and interrelationships between synovial fibroblast subsets in the lining and sub-lining layers of the synovium and observe how selective deletion of these subsets or changes in their biology alter the balance between persistent inflammation and tissue damage during the development of arthritis. Finally we will determine the relationship between alterations in fibroblast subsets and disease outcome during the development of human rheumatoid arthritis. A key strength of our work is that it combines both human and animal studies that run in parallel. An advantage of this approach is that we will minimize over reliance on mouse models of disease. This will place us in an ideal position to translate our findings into benefits for patients with inflammatory arthritis in general and rheumatoid arthritis in particular. Our studies represent an important step forward in the treatment of Immune Mediated Inflammatory Diseases as they allow the identification and therapeutic manipulation of discrete subsets of fibroblasts with the hope of modulating inflammation and cartilage/bone damage in the absence of immune suppression


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Foxa1/A2, Shh and Gli3 in T lymphocyte development
Funder: Medical Research Council

Abstract: We recently showed that conditional deletion of both the pioneer transcription factors Foxa1 and Foxa2 from thymic epithelial cells (TEC) had a profound influence on T-cell development, by reducing positive selection to the CD4+ T-lineage, while increasing production of regulatory T-cells. This project will investigate the way in which Foxa1 and Foxa2, the morphogen Sonic Hedgehog (Shh), and the Hedgehog (Hh)-responsive transcription factor Gli3, expressed by TEC, interact to control T-cell differentiation and the maturation of regulatory T-cells. We will investigate this using mouse Foxa1/Foxa2 and Hedgehog pathway conditional knockouts, mutants and reporter mice, in combination with in vitro culture systems for T-cell development and Treg function, and in vivo models. We will first dissect the individual contributions of Foxa1 and Foxa2 to TEC and T-cell differentiation and Treg maturation. We will use RNA sequencing and functional experiments to investigate how Foxa1 and Foxa2 in TEC modulate T-cell differentiation and Treg maturation and test the hypothesis that it is by changing expression of their selecting ligands (MHC+peptide). We will test the the hypothesis that expression in TEC of Shh promotes Treg maturation but Gli3 inhibits it, define Shh and Gli3 target genes in TEC, and investigate Shh/Gli3-Foxa1/a2 crosstalk. In parallel, we will investigate expression and function of Shh, Gli3, Foxa1 and Foxa2 in human TEC and T-cell development, using culture of human thymus explants.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Understanding DNA transport by topo IV and gyrase to counter antimicrobial resistance
Funder: Medical Research Council

Abstract: DNA gyrase and topo IV change DNA topology to regulate DNA supercoiling and chromosome segregation and are the validated targets of clinically important antibacterial fluoroquinolones. The enzymes act by capturing a DNA helix (the so-called T- or transport segment) and crossing it through a transient break in a second DNA segment (the so-called G-segment) via the formation of a 'cleavage complex'. From our solving the first structure of a cleavage complex, that of Streptococcus pneumoniae topo IV with bound quinolones, much is now known about the DNA gate. By contrast, the nature of the T-segment DNA and how it is captured and transported has remained elusive. We have now solved a second key structure - that of a Transport DNA segment captured in the hole formed by dimerisation of the ATPase domains of S. pneumoniae topo IV- the first glimpse of this important intermediate and which reveals intriguing features. The captured DNA is kinked, has a intercalated nucleotide and is bound asymmetrically by positively-charged residues lining the cavity whose mutagenesis severely inhibits or abrogates catalysis by topo IV. In the proposed research, we address four major questions. First, we shall use FRET, genetic and X-ray structural approaches to establish what identifies a transported DNA and whether kinking/ATP intercalation are integral to binding in the topo IV annulus. To illuminate the disposition of transport and gate DNAs and how DNA is navigated through the enzyme, we aim to solve an X-ray or cryo-EM structure of the full complex. Using mutagenesis of the cavity, we will identify residues essential to topo IV (and gyrase) activity suggesting a stratgy to guide drug design targeting transport. Finally, we shall translate these approaches to topo IV/gyrase of the ESKAPE pathogen, Klebsiella pneumoniae. Addressing these gaps in knowledge will advance topoisomerase mechanism and open new opportunities for design of novel agents able to combat quinolone resistance.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Regulation of arthritis by butyrate-mediated activation of regulatory B cells
Funder: Medical Research Council

Abstract: Regulatory B (Breg) cells restrain the severity of inflammation associated with immunological-disorders and help to limit collateral damage following unresolved infections. They act by directly suppressing the proliferation of effector T cells and promoting the differentiation of regulatory T cells, as well as inhibiting pro-inflammatory cytokine secretion by innate immune cells. Although it is known that the cytokine interleukin-10 (IL10) is crucial for these functions, the lack of a Breg cell specific marker has hindered the detailed characterization, and potential clinical utility, of these cells. We have recently identified the aryl hydrocarbon receptor (AhR) as a critical transcription factor for the differentiation of IL-10+Bregs. We have shown that arthritic mice lacking AhR expression in B cells display a profound decrease in Bregs in both spleen and in Gut-Associated Lymphoid Tissues (GALT). We have discovered that amongst a wide panel of microbiota-derived metabolites tested, the short-chain fatty acid, and AhR ligand, butyrate suppresses arthritis in a B cell dependent manner. Considering these findings alongside previous data showing that AhR ligands are abundant in the gut we hypothesize that: butyrate produced by gut bacteria, by enhancing the activity of the transcription factor AhR in B cells, promotes the differentiation of Breg. Our aims are: to identify whether butyrate promotes the differentiation of AhR+Bregs which can suppress autoimmunity, and whether butyrate acts directly on B cells or via an intermediate cell or bacteria; to understand the molecular mechanisms by which AhR promotes regulatory B cell function and how this is modulated by butyrate treatment. These experiments will result in a deeper understanding of how anatomical environmental signals are crucial for the generation of Bregs, and the detailed study of the Breg transcriptome will lead to new ways to identify the Bregs and reveal the multiple ways they may inhibit diseases.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of Rab46 in immune-mediated inflammatory diseases
Funder: Medical Research Council

Abstract: Objective 1: To determine if nucleotide variations in the Rab46 gene (EFCAB4B) are associated with common IMIDs, we will analyse genomic data stored in the UK Biobank from patients with several candidate diseases. We will compare data from patients with rheumatoid arthritis, atherosclerosis and psoriasis to a non-diseased control group matched for age and sex. We will determine any associations between mutations/SNPs in EFCAB4B and susceptibility to one or all disease phenotypes. Objective 2: To identify associations of Rab46 gene (EFCAB4B) with patient of non-clonal Mast Cell Activation Syndrome (ncMCAS) of unknown aetiology. We have a cohort of patients that are non-responders to omalizumab who exhibit hypersensitivity to mast cell degranulation. After collection of patient samples, we will prepare DNA libraries and whole genome sequencing will be performed using next generation sequencing facilities (NextSeq 500) based at the Leeds Institute of Molecular Medicine. Analysis will be performed by 'Leeds Omics' and focussed on Rab46 but the genomic sequencing data will be available for further GWA studies. Objective 3: The overarching strategy of this study is to use super-resolution imaging techniques (iSIM and AiryScan) to quantify and measure the co-localisation of distinct populations of granules with Rab46 in human mast cells or mast cells extracted from mouse bone marrow. Cells will be stimulated with IgE, ATP, C3a and TLR ligands and granule dynamics observed in fixed and live cells. The role of Rab46 in mast cell degranulation will be assessed biochemically using cells extracted from a global Rab46 knockout mouse (currently validated and maintained in the McKeown lab) as compared to wild-type littermates.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Back to basics; the role of complement C3 and its degradation fragments in immuno-inflammatory disease
Funder: Medical Research Council

Abstract: We will establish a collaborative network of experts in complement dysregulation, each focussed on different diseases and organs. Traditionally, the role of complement in inflammatory disease has been assigned to the central activation fragment, C3b, which is the driving force behind the complement amplification mechanism. However, recent data in genetically-modified animals, and genetic associations in man, have implicated downstream C3 degradation products in pathogenic processes. In this proposal, novel recombinant antibodies will be used in well-characterised cohorts of patients with diseases of different organs: the eye, brain and kidney. The antibodies will be used for three purposes: 1. To measure precisely levels of different C3 fragments in plasma and extend the biomarker dataset associated with established biobanks of samples and patient cohorts. Findings will be correlated with the extensive genetic and clinical phenotypic data already available for these collections. 2. To stain sections from the three organs for different C3 fragments, obtaining information regarding cellular location and leukocyte engagement. The diseases of focus in this pilot study are C3 glomerulopathy (kidney), age-related macular degeneration (eye) and multiple sclerosis (brain). 3. To carry out in vitro functional assays to correlate the biomarker data with genotypic and phenotypic data observed in patients with specific complement gene mutations. We aim to identify common mechanisms across a number of inflammatory diseases in diverse organs involving dysregulation of the complement system. The unique research tools described here will be used to validate a mechanism in sufficient depth to warrant a large scale follow-up proposal to confirm and explore common pathogenic processes utilising well-validated cohorts of patients.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Determining Regulatory Pathways That Maintain FOXP3 in Human T regulatory Cells
Funder: Medical Research Council

Abstract: CD4+ T-regulatory cells (Tregs) are important in maintaining immune tolerance and are characterised by Foxp3 expression. In particular, Tregs are efficient at inhibiting autoimmune diseases but regulatory processes that determine Foxp3 expression and stability in human Tregs is yet to be fully understood. My research on Treg biology has defined important molecular mechanisms that can maintain Treg cell function. Recently, I have uncovered a previously unknown regulatory mechanism involving post-translational regulation of Foxp3 in murine Treg cells. We found that Foxp3 can be degraded by a protease known as asparaginyl endopeptidase (AEP) in murine Tregs and PD-1 receptor signalling can significantly inhibit this process and maintain Foxp3 expression and stabilise Treg cell phenotype. Although previously AEP gene expression has been noted in Tregs, my work is the first to identify AEP protein, enzyme activity and functional role in murine Tregs. I will extend this work to study the importance of AEP in human Tregs and T helper cell subsets. The major goal of this project is to understand (a) the importance of AEP in human Treg cell function (b) identify coreceptor driven signalling pathways that induce AEP in human Tregs and (c) determine the function of AEP in human Th17 helper cell subsets. These research questions will provide a new in-sight into the post-translational regulation of human Tregs and demonstrate the role of proteases in regulating Treg and Th17 cell function.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Macrophage sensing of extracellular ATP during inflammation
Funder: Medical Research Council

Abstract: Innate immune recognition of danger signals is an important mechanism that protects against infection, and injury. ATP is released from infected or damaged cells and its extracellular presence alerts macrophages of impending danger. At high levels, extracellular ATP (eATP) binds to the P2X7 receptor, expressed on macrophages, triggering a potent pro-inflammatory immune response. This mechanism has been shown to be crucial in our defence against pathogens, but also plays a role in inflammatory diseases. Little is known about how P2X7R relays information from ATP binding, yet there is increasing evidence that kinase signalling plays an important downstream role, suggesting that the posttranslational modification phosphorylation might be key in this process. In this project, we will use quantitative proteomics to characterise the phosphorylation signals regulated by P2X7R upon eATP activation in macrophages. We will use this data to build computational network models driving hypotheses as to how these signals can be therapeutically targeted. We will also integrate how these networks control the biological outputs induced by P2X7R, for example release of inflammatory cytokines. Together, this will provide, for the first time, a comprehensive overview of the signalling triggered by the danger signal eATP in macrophages, providing avenues of potential new therapeutics in a wide-range of inflammatory diseases.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Epithelial micro-invasion and the inflammatory response to colonisation by Streptococcus pneumoniae in health and in vulnerable populations
Funder: Medical Research Council

Abstract: Using a novel pneumococcal controlled human infection model (PCHIM) we have recently discovered that during commensal carriage, S. pneumoniae invades the human mucosa triggering an epithelial innate-inflammatory response without causing disease. Transcriptomic analysis of the epithelial response shows that upregulation of inflammatory gene pathways is greatest around the time of pneumococcal clearance. We therefore hypothesise that this so-called micro-invasion of the mucosal epithelium and the subsequent innate-inflammatory profile determine the outcome of colonisation; and that in older adults, pneumococcal micro-invasion is enhanced and the innate-inflammatory response dysregulated, resulting in impaired clearance. We will now determine the role of the pneumococcal polysaccharide capsule and key sub-capsular components in micro-invasion using well-characterised epithelial cell systems. We will then identify the host gene expression pathways that characterise micro-invasion, discovering how pneumococcal micro-invasion is sensed in the epithelial cytosol. We will then use the PCHIM to detail the functional relationship between micro-invasive phenotype, the epithelial innate-inflammatory response and bacterial clearance. Finally, we will test whether pneumococcal micro-invasion is enhanced during colonisation in older adults and that in the context of so-called inflammaging, is associated with a dysregulated innate-inflammatory response and impaired clearance. Overall, this project will generate a fundamental understanding of the relationship between micro-invasion, the innate-inflammatory response and bacterial clearance. We will identify protective epithelial transcriptomic profiles associated with bacterial clearance that could be used for vaccine evaluation and optimisation. We will delineate the host-pathogen profiles that explain the vulnerability of older people to pneumococcal disease, and identify pathways for intervention.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: New insights into NLRP3 within inflammatory disease
Funder: Medical Research Council

Abstract: Inflammation is an early and important response to tissue injury and infection. In the absence of pathogens, it is considered sterile and exacerbates the pathology of major non-communicable diseases. Furthermore, the impact of co- or multi-morbidities is through an exacerbated inflammatory response. Inflammatory cytokines associated with damaging inflammation are commonly members of the interleukin-1 (IL-1) family. The pro-inflammatory IL-1 cytokines are regulated by the inflammasomes. Inflammasomes are cytosolic multimeric protein complexes formed in inflammatory cells in response to pathogenic infection and tissue injury. Activated inflammasomes drive the processing of pro-IL-1b to its mature secreted form. The most commonly studied inflammasome, and inflammasome most associated with disease, is composed of the cytosolic pattern recognition receptor (PRR) NACHT, LRR and PYD domains-containing protein 3 (NLRP3). NLRP3-dependent inflammation is specifically associated with the worsening of non-communicable diseases including atherosclerosis, Alzheimer's disease, type-II diabetes, arthritis and many others, so a research priority has been to understand the mechanism of NLRP3 inflammasome activation. Despite this our understanding of the regulation of the NLRP3 inflammasome remains incomplete. In part this limitation has been because of the lack of specific high level cell biology expertise. I aim to address this here combining and harnessing leading cell biology and immunology expertise to answer the biggest open questions in the inflammasome field: 1. How and where in the cell is the NLRP3 inflammasome activated (specifically defining disrupted endosomal trafficking as the unifying trigger for NLRP3 inflammasome activation)? 2. How is the NLRP3 inflammasome deactivated and removed (specifically understanding the role of autophagy in NLRP3 dependent responses)?


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