NIHRDataInsights/HRCSData huggingface dataset:
Title: Deciphering the effects of genetic variability in regulatory T cells - links with autoimmunity
Funder: Medical Research Council

Abstract: Aims: The majority of the causal variants underlying associations to complex traits remain unknown. This proposal aims to define the causal variants at loci associated with autoimmune diseases, by studying the effects of of associated variants on CD4+ regulatory T cells (Tregs). Objectives: Specifically, I will 1) construct a catalogue of chromatin and gene activity in Tregs from 100 individuals, 2) functionally prioritise putative causal variants based on their effects on gene regulatory features, as well as 3) gene and cytokine expression levels and 4) assess if the observed effects are specific cellular states, resting or stimulated. Methodology: Dr Trynka's group is generating functional genomic data from Tregs from 100 healthy blood donors. The assays include: 1) genome wide genotyping, 2) characterising active promoters by ChIP-seq for H3K4me3 histone modification , 3) mapping open chromatin regions using ATAC-seq, and 4) quantifying gene expression with RNA-seq. Assays 2-4 include both resting and stimulated Tregs. Using these datasets I will identify genetic variants that co-localise with H3K4me3 and ATAC features and test for correlation between alleles and the number of mapped sequence reads. Variants with allele-specific effects on histone modification levels or chromatin accessibility will comprise a pool of prioritised causal variants. I will further correlate these alleles with gene expression levels and assess the genetic effects on cytokine levels. Finally, if a subset of nominated causal alleles point to disrupted transcription factor binding (TF), I will perform functional validation using TF-specific ChIP-seq. Scientific and medical opportunities: We will gain insights into the causal molecular mechanisms underlying the immune disease loci that may lead to identification of novel drug targets and disease biomarkers. Finally, the framework proposed here can be easily adapted to other cell types and complex traits.


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 skeletal development and the use of anabolic agents in murine models of Duchenne muscular dystrophy
Funder: Medical Research Council

Abstract: Short stature and osteoporosis are common in Duchenne Muscular Dystrophy (DMD) and its pathophsyiology may include a paracrine as well as an endocrine abnormality of the GH/IGF-1 axis, which is further exacerbated by glucocorticoid (GC) use. The commonly used mdx mouse has a phenotype which is less severe than DMD boys. A new Cmah-/-mdx mouse model has increased disease severity, but its skeletal phenotype has never been investigated. To test the overall hypothesis that murine models of DMD have an intrinsic abnormality of linear growth and skeletal development that can be rescued by modulation of the GH/IGF-1 axis, I will investigate:- 1. Growth and growth plate (GP) chondrogenesis in mdx and Cmah-/-mdx murine models,expecting impairment in both, and increased severity in Cmah-/-mdx. A cross-sectional study of mice culled at 2,4 and 6 wks will be performed to determine linear bone growth rate at the chondro-osseus junction. Chondrocyte hypertrophy and proliferation will be quantified using image analysis and bromodeoxyuridine uptake. GP cartilage matrix production and GH/IGF receptor, IGF1,2 and IGFBP3 expression will be confirmed by immunohistochemistry. Metatarsal growth in explant culture will also be studied to examine any intrinsic skeletal abnormality. 2. Bone development and fragility in the Mdx and Cmah-/-mdx. A cross-sectional study of bone quality by micro-CT will be performed and the bone marrow studied to understand adipogenesis and osteoblastogenesis. 3. Growth response and bone development following exposure to GC,expecting them to be further reduced by daily dexamethasone. 4. Growth and bone response following treatment with rhGH and/or rhIGF-1, expecting combination therapy to be most effective. If rhGH or rhIGF-1 rescue bone and growth pre-clinically then this could be translated clinically. These data may also help inform the design of novel therapies directed specifically at the mechanism of insult leading to skeletal fragility in DMD.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of IKZF3 in B-cell differentiation and the pathogenesis of autoimmunity
Funder: Medical Research Council

Abstract: 1&2) Identify a panel of differentially expressed targets for Aiolos binding in B-cells during plasma-cell differentiation Chromatin immunoprecipitation, using an antibody currently being used for ChIP-Seq in a number of different cell types within the host laboratory, will be performed to identify target genes regulated by IKZF3 at Day 0, Day 3, Day 6, Day 13 and Day 20 of the plasma cell differentiation protocol. Differential expression of IKZF3 targets from RNA-Seq data will be assessed using an analysis pipeline currently in use within the host laboratory. Western blotting of the proteins coded by differentially-regulated expression targets will be used to verify the corresponding changes in protein expression for the differentially regulated IKZF3 target genes 3) Study the effect of IKZF3 knock-down on the differentiation of B-cells and expression of target genes IKZF3 expression in naïve B cells will also be knocked-down, using an siRNA used successfully in the host laboratory, to see how IKZF3 deficiency affects the numbers of cells, the ChIP-Seq peaks and RNA-Seq expression during the five stages of the plasma cell differentiation protocol from Aim 2. 4) Identify IKZF3-dependent biological pathways important in plasma cell differentiation. Having used bioinformatics techniques to correlate the DNA-binding affinity of IKZF3 with differential expression of gene targets (Aims 2-3), I will use Ingenuity Pathway Analysis to identify cell-type specific biological pathways which maybe important at each stage of plasma cell differentiation. Manipulation of the individual proteins in each of these pathways may yield potential regulators of plasma cell differentiation and be the subjects of my future research and which may lead to therapy.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Elucidating clock control of glucocorticoid action
Funder: Medical Research Council

Abstract: My host laboratory has recently discovered that the circadian clock regulates glucocorticoid action in the liver. Two candidate mechanisms have emerged, one involving a core clock protein, Cryptochrome (Cry), and the other a clock-controlled gene, Metadherin (MTDH). Both Cryptochrome and Metadherin interact with the glucocorticoid receptor (GR), and exert complex effects on GR biology. I will determine how these two candidates convey timing information to GR using mice lacking the two Cryptochromes (Cry1, Cry2), and mice lacking Metadherin. I will then investigate whether my findings are therapeutically tractable, in order to reduce the hyperglycaemia, hypertriglyceridaemia, and hepatosteatosis associated with glucocorticoid medication. In the initial experiments, I will examine acute effects of glucocorticoid on liver gene expression, by time of day, in Cry1/2 double knockout mice, in order to determine whether rhythmic repression of glucocorticoid transactivation effects, by Cry, contributes to circadian regulation of glucocorticoid action. I will test whether pharmacological augmentation of Cry can reduce glucocorticoid transactivation effects. I will then investigate GR regulation by MTDH by measuring the acute effects of glucocorticoid on liver gene expression and carbohydrate metabolism, by time of day, in MTDH knockout mice, compared to controls. The underlying mechanism will be defined by profiling gene expression globally using RNA-seq, and effects on GR function determined by ChIP-seq for the receptor in liver. I will investigate the convergence of pathways by measuring MTDH expression in Cry1/2 null mice, and by comparing my RNA-seq and GR ChIP-seq data with existing datasets, including Cry ChIP-seq, from wild-type liver. Finally, I will examine whether these candidate mechanisms can be targeted to ameliorate the effects of chronic glucocorticoid exposure, specifically the effects on glucose metabolism, triglyceride levels, and hepatosteatosis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Educating Macrophages in vivo
Funder: Medical Research Council

Abstract: We will identify the signals driving RELMa+ Arginase-1+ Ym1+ M2-like macrophage populations in vivo in a complex tissue environment, and to address the function of the intestinal macrophage. This can only be addressed in vivo where an inflamed physiologically relevant environment can be provided. We use Trichuris muris in the mouse, as an in vivo model of resolving gut inflammation. We have established this system as a robust tool for defining intestinal macrophage populations, including the emergence of waves of M1, and then RELMa+ Arginase-1+ Ym1+ M2-like macrophages. We combine this tissue inflammation model with cell-type specific gene-targeting mouse methodology. We use the CX3CR1Cre.IL4Rflfl mouse and the CX3CR1Cre.IL10Rflfl mouse to identify whether RELMa+ Arginase-1+ Ym1+ macrophages fail to emerge if the macrophage, in an environment where multiple stimuli are present, cannot receive a signal from IL-4/IL-13 or IL-10. We use an iNOS legacy mouse to identify in vivo whether RELMa+ Arginase-1+ Ym1+ macrophages transit through an M1 activation state, and whether this is essential for their emergence. We use multi-colour flow cytometry, and immunohistochemical approaches to identify macrophage subpopulations and other leukocytes in the gut (Little et al 2014 Journal of Immunology). We will identify the in vivo function of the intestinal macrophage by analysing (1) the reparative processes in the gut post T. muris infection, (2) the response to second infection and (3) the response to an inflammatory driver (DSS), in the presence of absence of the intestinal macrophage. We believe that there is an unmet scientific need to improve our understanding of macrophage biology in vivo; that our proposal will allow testing of in vitro paradigms in in vivo settings and that understanding macrophage behaviour in vivo is an essential prerequisite for the management of tissue inflammation, preluding the design of new immune-therapies to regulate/prevent chronic inflammation


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The inflammatory microenvironment of the post-pregnancy mammary gland and its role in tumourigenesis
Funder: Medical Research Council

Abstract: After pregnancy, the mammary gland undergoes extensive remodelling whereby milk-producing epithelial cells are replaced by adipocytes to return the gland to its pre-pregnant state. This regenerative process (involution) is associated with tumourigenesis and is thought to contribute significantly to pregnancy-associated breast cancer. Whilst the physiological processes of involution are well defined, the immunological implications are less well understood and are likely to be of great importance. Furthermore, the tumourigenic potential of involution is thought to increase with age and thus is likely to have an increasing impact in a society where women delay child birth until later in life. We want to investigate the role of the immune system in involution to determine how the mammary microenvironment becomes tumourigenic and how this is exacerbated in older women. To fulfil these aims, we have an armoury of mechanistic in vivo models which have been established during our current MRC funding. We have already identified many of the leukocyte populations present in the gland during involution. To fully characterise these cells we will interrogate their molecular function, by ex vivo stimulation of isolated cells and subsequent cytokine analysis. Our previous microarray highlighted key players in this respect. To identify the role of these mediators in involution we will use genetically altered mice which lack these components in conjunction with depletion of specific cell types, namely macrophages. We will then utilise our established in vivo syngeneic tumour model to determine firstly how involution contributes to tumourigenesis, and secondly how the molecular mediators outlined above contribute to tumourigenesis. Finally, using cohorts of aged mice we will dissect how age affects involution-mediated tumour development.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Tissue-specific T cell migration in the intestine
Funder: Medical Research Council

Abstract: The small intestine (SI) and colon are physiologically and functionally distinct. Adaptive immune responses in the intestine are initiated in the mesenteric lymph node (MLN) chain. We have found that the individual MLN are immunologically distinct. Dendritic cells (DCs) and lymph from the SI drains to specific small intestinal nodes (sMLN), while DCs and fluid from the colon enter colonic nodes (cMLN). T cells activated in the sMLN can express CCR9 and alpha4beta7 and preferentially home to the SI, and we have shown that T cells in the cMLN selectively migrate to the colon. This separation of sMLN and cMLN provides a mechanism by which immune responses in the SI and colon can be independently controlled. Even greater regional specialisation may exist because CCR9-dependence varies along the SI, and we find that DCs from particular segments of the SI migrate to specific sMLNs. Drugs targeting CCR9 and alpha4beta7 have been developed to treat inflammatory bowel disease (IBD). Further insights into the molecular mechanisms underpinning tissue-specific homing will likely provide future targets for drug development. We hypothesise that specific nodes in the MLN chain imprint distinct molecular signatures on activated T cells to allow their preferential homing to precise regions of the intestine. We aim to define, with unprecedented precision, how trafficking of T cells activated in different MLNs is controlled; the molecular signals directing T cell migration to parts the intestine; and the cells and molecules that participate in imprinting these migratory preferences. To fully understand the physiological relevance of these processes, we will perform our experiments both in the steady state, and in animals with small intestinal or colonic inflammation. This study will enhance understanding of critically important immunological processes, and identify novel targets whose therapeutic manipulation could enhance mucosal vaccination or provide new therapies for IBD.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Sampling the environment: Antigen presentation and T cell regulation by intestinal epithelial cells
Funder: Medical Research Council

Abstract: The aim of the project is to define the response of intestinal epithelial cells (IEC) during intestinal inflammation and to assess how this impacts on intestinal immune regulation. This is of special interest in the context of inflammatory bowel disease (IBD), were regulatory pathways fail and the immune system drives a pathogenic T cell response towards commensal bacteria. Our preliminary data indicate that the antigen processing and presentation pathway is the most highly enriched pathway in IEC during colitic conditions. Therefore, we aim to assess the functional consequences of MHCII antigen presentation by IEC on the immune cell compartment during homeostasis and during conditions of intestinal infection and inflammation. This will be achieved by generating transgenic mice in which MHCII expression is selectively ablated in IEC through the lineage-specific expression of the Cre recombinase. The CD4+ T cell compartments of these mice will be analyzed during homeostasis and in models of bacterially-triggered intestinal inflammation that reproduce many aspects of the inflammatory pathology associated with human IBD. These analyses will be complemented by using primary IEC organoids as APC in a co-culture system with ova-specific CD4+ T cells (OTII cells). We will use this ex vivo system to address the antigen processing and presentation capabilities of IEC as well as the impact it has on the activation, proliferation and differentiation of CD4+ T cells. Furthermore, to confirm the validity of our results in a human system, primary IEC organoids will be generated from healthy or IBD patients and analysed for their antigen processing and presentation capabilities, as well as their responses to inflammatory stimuli. A better understanding of the crosstalk between IEC and T cells in the intestinal mucosa may lead to the development of novel therapies for IBD.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: BIOlogical Factors that Limit sustAined Remission in rhEumatoid arthritis (the BIO-FLARE study)
Funder: Medical Research Council

Abstract: Flares of immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA), are a major burden for patients. They occur unpredictably, adding to the physical and psychological burden of the condition, and contribute to joint damage and comorbidities, such as cardiovascular disease. Flares are difficult to study, precisely because of their unpredictable timing. However, we have established a human model that generates a 'synchronized' population of RA patients in clinical remission, of whom about 50% flare within 6 months, the remainder maintaining remission. We propose to carefully study such a population to better understand the factors that determine disease flare. Up to a third of patients with RA achieve disease remission with aggressive therapy. Many wish to discontinue therapy at this point and, if they do, about 50% flare in the subsequent 6 months. We will follow such a population prospectively, to study the biological processes underpinning flare. We will study 150 patients in clinical remission for at least 6 months, and prepared to discontinue treatment. After treatment cessation patients will be studied intensively, with blood sampling at weeks 2, 4, 6, 12 and 24. Patients will also undergo ultrasound-guided biopsy of 'flaring' synovium before reinstatement of previous treatment (with which patients readily regain remission). We will also obtain biopsies from consenting patients at baseline and in remission at 6 months. In blood we will study immune cell subsets and their activation status; autoantibody profiles; and epigenetic profiles. Synovial biopsies will allow us to examine the cell lineages present, including stromal cell subtypes, as well as inflammatory mediators. We will additionally examine metabolic profiles in blood, urine and synoivum. Using multivariate statistical techniques we will develop a model that predicts impending flare in RA, highlighting tractable therapeutic pathways and informative biomarkers.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Understanding VRAC-dependent regulation of the NLRP3 inflammasome
Funder: Medical Research Council

Abstract: Inflammation that occurs in response to tissue injury or disease can contribute to damage. Members of the interleukin-1 (IL-1) family of cytokines are often implicated in disease processes, namely the pro-inflammatory members 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 pathogen or damage associated molecular patterns (PAMPs or DAMPs) that stimulate pattern recognition receptors (PRRs) on cells of the innate immune system. Once expressed, pro-IL-1 remains intracellular until an additional PAMP or DAMP stimulation activates inflammasomes. Inflammasomes activate caspase-1 which in turn processes pro-IL-1 allowing release of the mature cytokine. The inflammasome most implicated in non-communicable diseases is formed by the PRR NLRP3. The NLRP3 inflammasome is also the best studied inflammasome, yet there are still large gaps in knowledge concerning its activation. We have discovered that the volume-regulated anion channel (VRAC), a Cl- channel composed of LRRC8 subunits, could be an important regulator of the NLRP3 inflammasome. We now aim to verify the importance of VRAC to NLRP3-dependent inflammatory responses using a number of CRISPR based strategies to knockout the VRAC essential sub-unit LRRC8A. Knocking out LRRC8A in vitro will allow us to verify the importance of VRAC in NLRP3 inflammasome activation in response to a range of well-characterised NLRP3 activating stimuli. We will also knock out LRRC8A in macrophages specifically and demonstrate its relevance in an in vivo model of NLRP3-dependent inflammation. This, combined with our repurposing of existing medicines as VRAC inhibitors, will establish VRAC as a potential novel therapeutic target for the treatment of inflammatory disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Hedgehog signalling in peripheral T cell differentiation and function
Funder: Medical Research Council

Abstract: This project will investigate the roles of the Hedgehog (Hh) signalling pathway in peripheral T cell differentiation and function, and the molecular mechanisms that underlie these functions. We aim to understand how Hh proteins produced by peripheral tissues influence T cell fate. We will investigate Hh function in peripheral T cells in mouse Hh pathway mutants and conditional knockouts, TCR-transgenics, and Hh- and TCR-reporter mice, using in vitro assays of T cell activation and function, and in vivo models, analysing by flow cytometry, microscopy, protein chemistry, transriptomics, and ChIP. We will use TCR-reporter transgenics and Hh-reporter transgenics to test the hypothesis that Hh pathway activation reduces TCR signal transduction at the single cell level, and to investigate cross-talk between Hh and TCR signal transduction. We will define the different functions of the three Hedgehog proteins in CD4 and CD8 T cell differentiation and test the hypothesis that Hedgehog pathway activation influences Th17 induction. We will test the impact of manipulation of Hh signalling to T cells on tumour rejection. We will use RNAseq and ChIPseq to identify Hh target genes in CD4 and CD8 cells and we will investigate the function of selected target genes in T cell differentiation and effector function.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM