NIHRDataInsights/HRCSData huggingface dataset:
Title: Investigating lactate as an inflammatory early signal in ectopic lymphoid neogenesis and its translational impact in patients with autoimmune diseases
Funder: Medical Research Council

Abstract: Based on our published and new data (revised manuscript resubmitted at Cell Metabolism), we hypothesize that T cells entering the inflamed tissue sense lactate via the expression of SLC5A12 with consequent loss of motility, increased retention at the site, and enhanced production of IL-17. Thus, we will test that: i) CD4+ T lymphocytes infiltrating the inflamed tissue undergo a profound intracellular metabolic rewiring and activate biosynthetic pathways as a consequence of lactate influx through the carrier SLC5A12, which in turn causes the observed marked production of IL-17 and reduced migratory abilities by CD4+ T cells; ii) This is a fundamental process in the control of ELS formation, maintenance and function in the inflamed tissue. We plan to proceed in 3 progressive steps performing a comprehensive set of ex vivo, in vivo and in vitro experiments, as reflected in the plan of investigation: Step 1) Investigate SLC5A12 and IL-17 expression on peripheral (blood) and salivary gland CD4+ T-cells in ELS+ versus ELS- SS patients and before/after Rituximab: i) Multicolour confocal microscopy coupled to digital image analysis; ii) Multicolour FACS analysis; iii) Transcriptomic analysis. Step 2) Assess the impact of knocking out SLC5A12 (slc5a12-/- mice) and the therapeutic potential of blocking SLC5A12 in modulating ELS formation, maintenance and function in an inducible murine model of salivary gland ELS that we have developed and in SS salivary gland organ cultures: i) Murine model of adenovirus-induced salivary gland ELS; ii) Ex vivo salivary gland organ cultures. Step 3) Dissect the metabolic and intracellular pathways responsible for lactate/SLC5A12-mediated induction of IL-17 and other inflammatory cytokines and inhibition of migration in CD4+ T-cells from SS patients: i) Mass spectrometry unsupervised metabolomics; ii) Mass spectrometry stable isotope (13C-lactate) tracer analysis; iii) Pathway/functional analysis


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Developing temperature and osmolarity based therapeutic approaches to modulate circadian rhythm in degenerative intervertebral disc and promote repair
Funder: Medical Research Council

Abstract: Circadian clocks drive ~24 hour rhythms in nearly all aspects of our physiology and behaviour. Changes of circadian rhythms during ageing underpin many of the age-associated pathologies. Low back pain is amongst the most prevalent spinal diseases associated with increasing age. It causes severe pain and loss of mobility, with currently no long-term cure. Progressive degeneration of the intervertebral disc (IVD) tissue is a major contributing factor. However, how susceptibility to IVD degeneration and LBP increases with age is poorly understood. Funded by the MRC, our ground-breaking work has demonstrated cell autonomous circadian clocks in mouse and human IVD. The IVD circadian clock controls rhythmic expression of >600 genes, many are known key players in IVD tissue homeostasis. Genetic disruption of the intrinsic IVD clock in mice leads to tissue damage characteristic of human disc degeneration (fibrosis and ossification). Building on these exciting discoveries, we now aim to combine our unique IVD-"clockless" transgenic mouse, ex vivo IVD explant and clinical IVD samples to establish how circadian clock disruption leads to IVD degeneration and whether we can use pharmacological compounds that target clock genes, or other approaches (such as periodic heat pulses or osmotic mimetics) to entrain circadian rhythms in degenerative IVD and thereby promote tissue repair. This research holds strong potential to identify novel therapeutic strategies for low back pain, which will have a long lasting impact on the health and well-being of the ageing population. This is particularly important because demographic ageing and the modern 24/7 life style frequently disrupt our circadian rhythms which can significantly increase risk of many diseases, including low back pain.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Optimising therapeutic strategy in psoriasis
Funder: Medical Research Council

Abstract: Psoriasis affects 2-4% of the population and NHS costs are >£750 million/year, largely due to biologics costing £10,000/year/patient. NICE recommend 10 biologics targeting TNF or IL-23/T17 cytokines with equal first-line weighting. Real-world data suggest 20-30% of patients fail to respond, highlighting inter-individual variation in driver immune pathways. We thus urgently need to understand which biologic best suits each patient. Whereas biomarkers guide cancer care, such strategies are lacking in inflammatory diseases. Given research by the PSORT biomarker discovery programme, psoriasis presents an ideal disease model for this work. Hypothesis: An algorithm which stratifies patients to receive TNF or IL-23/T17 inhibitors on the basis of genetic and clinical biomarkers, followed by dose optimisation using therapeutic drug monitoring (TDM), will improve real-world outcomes. Aims: 1. Develop a treatment algorithm to personalise biologic use 2. Evaluate the algorithm's clinical utility in silico using a multicentre real-world observational pharmacovigilance dataset 3. Design a prospective biomarker-strategy trial to assess the algorithm against standard care Methods: I will develop an algorithm, using PSORT's existing dataset and logistic regression, that stratifies patients to receive TNF or IL-23/T17 inhibitors based on established genetic (HLA-C*06:02) and clinical (e.g. psoriatic arthritis) biomarkers of response. TDM will prompt dose modification in those with supra/subtherapeutic drug levels. The algorithm's clinical utility will be determined in silico in an existing independent longitudinal outcome bioresource containing genetic, clinical and drug level data on patients receiving biologics, using area under the ROC curve. A trial will be designed to assess the algorithm. By personalising treatment selection, this study will improve outcomes and cost efficiency of care and provide a blueprint for the management of other inflammatory diseases.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Modulating Metabolic Programming of Memory B cell Activation to Restore B-cell Homeostasis in Autoimmune Disease
Funder: Medical Research Council

Abstract: A novel BCR-AMPK-Sestrin regulatory pathway of B-cell activation Upon encounter with specific antigen, the B-cell receptor (BCR) directs naïve B cells to differentiate into memory B cells (MBC) and antibody secreting cells (ASCs). Antigen re-challenge results in disparate response in memory B cells such that some become activated rapidly whereas others remain quiescent. This disparity may be regulated by differential expression of key co-receptors such as CD19 and CD38. Immune defects may alter B-cell homeostasis in autoimmune disease such as Systemic Lupus Erythematosus (SLE). Further, antigen-challenge evokes a state of cellular stress, which may trigger the expression of stress-inducible proteins, Sestrins. Prof Akbar's group has demonstrated that Sestrins co-operate with AMPK, form a complex with p38 MAPK, Erk and Jnk referred to as Sestrin-p38MAPK complex (sMAC) to drive T cell senescence whilst inhibition of sMAC enhances vaccine responsiveness in old mice. However, their role in B cells is not known. My preliminary data developed in collaboration with Prof Akbar's team, revealed differential expression of phosphorylated-AMPK (p-AMPK), p38MAPK and sestrins in BCR-stimulated B-cell subpopulations correlating with IgD and CD38 expression in MBC whereas siRNA-mediated knock down of sestrin-2 was associated with significantly lower expression of IgD and CD38. In eight samples from patients with SLE, B cells with higher IgD expression had 2-4 fold higher expression of p-AMPK compared to those with lower IgD expression. Together these results suggest that the BCR recruits CD38 to influence MBC activation. Therefore, the proposal seeks to explore a novel sestrin-AMPK regulatory pathway of B-cell activation through a series of in vitro experiments using blood samples from healthy people and those with SLE toward Modulating Metabolic Programming of B cells to Restore B-cell Homeostasis in Autoimmune Disease such as SLE.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Precision genome editing with tandem autologous transplantation as a therapy for multiple severe immune-mediated diseases
Funder: Medical Research Council

Abstract: Autologous stem cell transplant (ASCT) is an effective treatment in immune-mediated disease (IMDs) because the combination of high dose chemotherapy and anti-thymocyte globulin transiently induces severe immunosuppression, which eradicates a large proportion of pathogenic cells. The stem cell transplant repopulates the immune system without these clones. Our approach is to combine ASCT with genome editing of haemopoietic stem cells (HSCs) to precisely change the genotype of bone marrow-derived immune cells to sequences that are highly protective against developing IMDs. This would represent a novel treatment modality that would potentially be applicable to a broad range of IMDs. GWAS and UK Biobank analyses have revealed naturally occurring protein coding variants in a target gene that are highly protective against developing a broad range of IMDs (odds ratios of 0.1-0.2). We plan to replicate the effects of these variants using the novel CRISPR-cas9 base editors to make precise edits in the protein coding sequence of the gene. Significant recent advances (particularly the adoption of modified guide RNAs and purified Cas9 protein) mean that we can now achieve >95% editing in primary human HSC without cell selection. In preliminary experiments we have also been able to achieve >85% editing with Cas9 base editors in primary human haemopoietic stem cells using plasmids. We anticipate it will be possible to achieve >95% editing in human HSCs in the near future using base editor ribonuclearprotein. This will open up new avenues of treatment for IMDs as it will allow us to make precise long-term changes to the immune system.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: How does blocking inflammation enhance human cutaneous immunity during ageing in vivo?
Funder: Medical Research Council

Abstract: Leukocyte populations will be isolated from 5mm punch biopsies taken from normal the skin of young (<35 years) and older humans (>65 years) and also from the site of injury after the injection of 20ul of air into the skin. These samples will be used for immunohistology coupled to confocal microscopy or will be digested and sorted by multiparametric flow cytometry. In addition we will perform single cell RNAseq analyses on cells isolated from skin biopsies with Prof Clatworthy in Cambridge. This data will be analysed further in collaboration with Muzlifah Haniffa's skin atlas to determine how different skin cell populations change with age. We will also use an experimental system where senescent and non-senescent dermal fibroblasts will be co cultured with autologous CD4+ T cells to determine if the inflammatory cytokines secreted can induce FoxP3 expression and regulatory function. We will test if the interaction between senescent fibroblasts and different autologous monocyte subsets from the blood can amplify the secretion of inflammatory mediators by either cell type. Another inhibitory mechanism identified in our preliminary RNAseq studies of skin biopsies from young and old subjects is PTGS2 which encodes the protein COX2, which is upregulated under inflammatory conditions and results in the production of the lipid mediator Prostaglandin E2 (PGE2). Preliminary analysis of skin sections has identified that COX2 is elevated in old saline-injected skin compared to normal skin taken from the same subjects. We will Investigate prostaglandin E2 (PGE2; COX2 pathway) production by infiltrating monocytes and whether the PGE2 receptors (EP4 and EP2) is increased on T resident memory (Trm) cells (identified by CD69 co-staining). Biobanked samples of normal and air/saline injected skin from old individuals before and after p38 inhibition in vivo will be interrogated by immunohistology and confocal microscopy for the cellular interactions described above.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MRC Transition Support CSF David Owen
Funder: Medical Research Council

Abstract: We have: Set up an immunology laboratory and dedicated iPSC facility Derived microglia-like cells from iPSC cultures Characterised the bioenergetics profile Demonstrated equivalence with primary microglia Identified the effect TSPO KO has on microglial bioenergetics and TNF-a production Identified novel TSPO binding partners We now need to demonstrate the molecular mechanism underlying how the interaction with the binding partners mediates the effect of TSPO on microglial bioenergetics and microglial immunophenotype. Aim 1 Complete the immunological characterisation of TSPO KO cells and show that expression of wildtype TSPO in TSPO KO cell lines rescues the immune phenotype. Aim 2 From our newly discovered binding partners, we are initially prioritising MTCH2 (see CfS to explain why). We will test the hypothesis that the effect of TSPO KO on the immunophenotype is mediated by the increased leak currents across the mitochondrial inner membrane, itself mediated by downregulation of MTCH2. We will therefore overexpress MTCH2 in TSPO KO cells to determine if this partially rescues both the bioenergetic changes (associated with the increased leak) and the immunophenotype. We expect that the changes in mitochondrial energetics, in general or by overexpression of MTCH2, mediate the immunomodulatory effects of TSPO. Modulation of the immune phenotype by mitochondria is likely to involve a molecular complex that interacts with TSPO. We will therefore also focus on HK1, as another player in this paradigm (again, see CfS to explain why). We will test the hypothesis that HK1 activity is directly reduced in TSPO KO, and that expression of wildtype TSPO in the TSPO KO restores HK1 activity and rescues the immune phenotype. If so, we will test the hypothesis that the effect of TSPO KO on the immunophenotype is mediated by a reduction in HK1 activity. We will therefore overexpress HK1 in TSPO KO cells to determine if this partially rescues the phenotype.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MRC Transition Support CSF Alexandra Santos
Funder: Medical Research Council

Abstract: The majority of children with food-specific IgE do not have food allergy (FA). In equivocal cases, oral food challenges (OFC) are required. However, these are expensive, time-consuming and can cause allergic reactions. Aims: 1. To improve the diagnosis of food allergy using the basophil activation test (BAT) and the mast cell activation test (MAT); 2. To understand the molecular and cellular basis underpinning FA and tolerance in IgE-sensitised children. Methodology: Following the success of BAT to peanut in diagnosing food allergy (97% accuracy and 66% reduction in OFC), we have developed the BAT to milk, egg, cashew and sesame and are currently assessing the diagnostic utility of BAT to diagnose allergy to these foods in the BAT2 Study. Children with suspected allergies to these foods have been invited to participate and BAT has been performed in real-time. MAT has been done in parallel as it has the advantage of providing results for subjects with non-responding basophils and of using stored rather than fresh blood samples, which allows to test samples collected far from the lab or in the past. The performance of BAT and MAT will be compared with the gold-standard OFC. In order to better understand why some children with food-specific IgE react to the food whereas others do not and also why some FA resolve whereas others tend to be persistent, we will be studying how different subsets of T cells respond to allergen in allergic versus non-allergic patients and in transient versus persistent food allergies. These experiments will provide the foundation for a future Senior Fellowship application. Scientific and medical opportunities: With the validation of the BAT and the MAT to diagnose common food allergies, we will improve the accuracy and the safety of food allergy diagnosis. By comparing the T cell response between patients and between allergens, we will identify potential targets for a definitive treatment of FA, which we currently do not have.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Epigenetic modifications in Dupuytren's diseases
Funder: Medical Research Council

Abstract: Despite the frequency of its occurrence (prevalence >7% in the USA), Dupuytren's Disease (DD) of the hand has received limited attention to date. DD pathogenesis likely involves several inflammatory and fibrotic molecular pathways that are already characterised in other contexts, and thus a broad array of potential targets for drug development and early diagnostic markers likely exist. As such, it provides a good example of the challenge faced by researchers attempting to unravel molecular mechanism in common diseases of underappreciated functional impact. While a helpful nonsurgical treatment for symptoms of the DD has recently become available, the molecular mechanisms that lead to its emergence are not understood. Our preliminary data suggest that an epigenetic modification driving aberrant tissue fibrosis offers a potential for therapeutic manipulation of DD. relevant disorders across broader clinical fibrosis domains. We propose a feasible programme of preclinical work using existing methodologies, preclinical tool compounds and laser capture methods. The readouts in vitro and ex vivo are clear and will allow a milestone driven approach as envisaged in EMINENT programmes. We propose this as a preclinical project that if successful has potential to identify new targets and may allow repositioning of existing drugs to treat DD.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Elucidating and modulating macrophage and stem cell responses to bioactive nanoclays for bone regeneration
Funder: Medical Research Council

Abstract: Nanoclay gels offer new possibilities for regenerative medicine given their ability to assemble gels, promote cellular ingrowth and bind biological molecules. By incorporating growth-factors such as BMP2, we have shown the potential to achieve bone induction at the lowest dose published in the literature to date. We propose to utilise this strategy in spinal fusion and fracture repair to induce bone formation with greater precision and safety. Immune cells such as macrophages, are key in mediating the colonisation of an implanted biomaterial. There is strong evidence to indicate that nanoclay gels are successfully colonised and remodelled by stem cells, but the mediating role of macrophages, particularly in relation to stem cell recruitment and nanoclay degradation and clearance remains unclear. Previous work by the Kyoto group has shown the potential of dual drug release strategies from hydrogels to achieve efficient macrophage polarisation and enhance regenerative responses. In this collaboration we will explore the early macrophage response to nanoclay gels and develop a drug release strategy utilising the peroxisome proliferator-activated receptor gamma agonist pioglitazone (PIO) - a well-known modulator of inflammatory responses currently licensed for treatment of type II diabetes - to explore the effect of macrophage polarisation on nanoclay hosted BMP2 bone induction. This work will build on a successful micelle based approach developed in Kyoto to solubilise pioglitazone for hydrogel incorporation and controlled release. In vitro studies will examine macrophage phagocytosis and signalling in response to nanoclay and characterise macrophage-stem cell cross talk. In vivo ectopic bone and calvarial defect assays will harness unique in vivo radiolabelling (Kyoto) and cell and clay tracking (Soton) capabilities to dissect the dynamics of drug release, clay degradation, macrophage and stem cell recruitment and bone induction.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Human secreted IgD: structure, interactions and mechanisms in allergic inflammation and asthma
Funder: Medical Research Council

Abstract: We will investigate the structures, interactions and functional mechanisms of secreted IgD antibodies. The research project seeks to identify and characterise the key molecules that regulate these processes. The project proposes five interconnected strands of work: 1. Development of a novel set of anti-IgD antibodies, selected for the ability to inhibit interactions between IgD and specific IgD ligands. These selective inhibitors will allow intervention and elucidation of interaction pathways that mediate IgD's pro-inflammatory effects. 2. Identification and characterisation of the cellular receptor(s) for IgD. 3. Characterisation of the molecular interactions between IgD antibodies and antigens and superantigens, receptors, and anti-IgD antibodies, using a suite of biophysical methods. 4. Determination of high-resolution three-dimensional structures for IgD and IgD complexes with receptors and various ligands, utilising an integrated structural biology approach. 5. Examination of the effector functions of antigen-specific IgD antibodies in cellular assays using well-defined recombinant proteins and cell lines, and in vitro studies using patient derived cells. These experiments will test specific hypotheses developed from the molecular studies to elucidate roles of IgD in allergic inflammation.


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