NIHRDataInsights/HRCSData huggingface dataset:
Title: Investigation of the generation and functional maturation of regulatory T cells in vivo
Funder: Medical Research Council

Abstract: This project aims to understand the mechanisms that control the generation and functional maturation of Treg during inflammation and upon antigen recognition in vivo. To this end, the project will investigate in vivo mechanisms for Foxp3-mediated T cell regulation. We will use our novel Fluorescent Timer approach (designated as the Tocky system, see Objectives and Case) to identify and analyse new Treg and effector Treg (eTreg) by their Foxp3 transcription dynamics, and analyse their molecular mechanisms. Although preceding studies identified the factors that activate Foxp3 transcription, the physiological significance of these factors in vivo is still obscure, mainly because, there had been no efficient method to analyse the in vivo dynamics of Foxp3 transcription before the recent development of our Tocky system. Because of this, the biological relevance of induced Treg and transient Foxp3 expression is obscure. However, the Tocky system allows the investigation of the in vivo dynamics of Foxp3 transcription and Treg generation under physiological conditions. In addition, using Foxp3-Tocky, eTreg can be effectively identified and analysed. In the proposed project, we will use the Tocky technology together with our novel double and triple transgenic mice, and thereby investigate the molecular mechanisms for how dynamically Foxp3 expression is regulated and how Foxp3 protein controls the homeostasis of Treg and eTreg differentiation in vivo.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: Modulation of IL-33-dependent responses using parasite products
Funder: Medical Research Council

Abstract: In this project, we will characterise modulation of IL-33-dependent eosinophilic or neutrophilic responses by HpARI, HpBARI (and derivatives of these), parasite-derived modulators of the IL-33 pathway. Objectives: 1. Show therapeutic efficacy of HpARI, HpARI_CCP1/2 and HpBARI in eosinophilic and inflammatory responses. HpARI, HpARI_CCP1/2 and HpBARI will be administered into mouse airways with Alternaria, LPS, HCl or S. pneumoniae. Mice will be randomised to experimental groups, and cage-blocked. Power calculations have been used to ensure statistical significance is attained at expected effect sizes. Inflammation and damage will be measured by histology, flow cytometry, and cytokine/chemokine measurements. Human IL-33 knock-in, mouse IL-33 knock-out mice, and culture of human PBMCs with recombinant cytokines and HpARI/HpBARI derivatives will be used to translate results to human systems. 2. Elucidate the molecular mechanism of action of HpARI/HpBARI, and design minimally immunogenic mimetics. Binding partner identification will be carried out using biotin label transfer and streptavidin pull-down, with targets identification by tandem mass spectrometry. Binding to murine and human targets will be characterised by SPR. Binding sites will be identified using HDX-MS. This will allow us to characterize the binding partners and molecular mechanism of action of HpARI and HpBARI, and to design humanized proteins, in which active sites are cloned into a host CCP protein scaffold (FBa). 3. Investigate the role of IL-33 in the recruitment of neutrophils. We will assess the role of the IL-33 pathway in models of acute lung injury (HCl administration) or pneumonia (S. pneumoniae infection). The IL-33 pathway will be blocked using ST2-KO mice, mAbs, or HpARI/HpBARI administration. We will assess the IL-33-mediated activation and cytokine production of innate immune cells, chemokine responses, and neutrophil chemokine receptor expression.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Unravelling the role of CD8+ T-cells in immune checkpoint inhibitor induced colitis
Funder: Medical Research Council

Abstract: Immune check point inhibitors (ICPIs) including anti-CTLA-4 (e.g. ipilimumab) and anti-PD-1 (e.g. nivolumab), have revolutionized cancer therapy. However, whilst augmenting T-cell activation with immune checkpoint blockade is effective in anti-tumour responses, it can also impair self-tolerance leading to toxicities. The most frequently occurring serious and life-threatening toxicity includes colitis, affecting up to 50% of patients, especially when the more efficacious combination regimens (anti-CTLA-4 and anti-PD-1) are used. This often necessitates ICPI discontinuation. Timely delivery of targeted anti-inflammatory therapy to reduce morbidity and mortality associated with colitis, and avert interruption of life extending treatment has been hampered by a lack of understanding of the immunopathological processes underlying ICPI-induced colitis. Whilst emerging data suggests excessive CD8+ activation drives gut inflammation in IBD and ICPI-colitis, the pathways mediating the response are unknown. To interrogate the pathways underlying ICPI induced colitis further, I will: 1) Define checkpoint molecule expression by mucosal CD8+ T-cells using multiparameter flow cytometry. This may shed insight into the possibility that differential expression of different regulatory pathways by mucosal CD8+ T-cell may underpin susceptibility to ICPI- colitis. 2) Define effector function and metabolic phenotype of blood and mucosal CD8+ T-cells following in vitro and in vivo exposure to ICPIs. 3) Use an epithelial organoid/CD8+ T-cell co-culture system to mechanistically probe immune/epithelial interactions, including the natural killer group 2D (NKG2D) pathway which is implicated in mediating mucosal gut inflammation in IBD. To mitigate against the possibility that CD8+ cells are not the key drivers of inflammation, I will capture data on other immune cells (e.g. CD4+ T-cells) and pathways.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The microbiome drives circadian rhythms in inflammation
Funder: Medical Research Council

Abstract: The circadian clock allows organisms to adapt their physiology and behavior to the 24h environment. This internal timer regulates many aspects of physiology, including tissue immune homeostasis. In chronic inflammation, clock oscillations persist within affected tissue, driving rhythmicity in inflammatory pathways. In mouse experimental arthritis (collagen induced arthritis) joint inflammatory pathways are repressed during the night. We propose that this results from the circadian influx of T regulatory cells (Tregs) at night. The immunosuppressive activity of Tregs is influenced by the gut microbiome, which itself is highly circadian rhythmic, with daily changes in bacterial numbers, mucosal proximity, and metabolite production. This project tests the role of microbiome-derived metabolites in conferring circadian behavior to joint Treg cells, and thereby establishing a link between feeding behavior and circadian control of inflammation. We will assess quantitative and qualitative changes in Tregs within inflamed joints at different phases of the day (flow cytometry, co-culture assays and RNA-Seq). Depletion of Tregs from mice with established inflammatory arthritis will test the hypothesis that Tregs drive diurnal rhythms in inflammation. To examine temporal changes in Treg trafficking we will utilize Kaede mice (in which Tregs can be photo-converted at specific anatomical sites) and adoptive transfer studies. The final workstream will assess the role of the gut microbiome on conferring time-of-day regulation to Treg function and trafficking. We will assess how feeding times and chronic inflammation affect the composition of the microbiome over the course of 24h and its and metabolic outputs (mouse and human). Finally, we will test whether dietary intervention can stabilise Treg activity over the course of 24h thereby improving disease outcome.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Treatment of inflammation via activation of the mRNA-destabilising protein tristetraprolin
Funder: Medical Research Council

Abstract: The mRNA-destabilising protein tristetraprolin (TTP) is a powerful negative regulator of many pro-inflammatory genes. This project tests the concept that the broad anti-inflammatory effects of TTP can be harnessed to achieve therapeutic effects in inflammatory diseases. Both the expression and activity of TTP are controlled by reversible phosphorylation. The inflammation-responsive kinase MK2 mediates phosphorylation and inactivation of TTP, increasing expression of inflammatory mediators. The phosphatase PP2A mediates dephosphorylation and activation of TTP, decreasing the expression of inflammatory mediators. We made a genetically modified mouse strain in which endogenous TTP protein cannot be phosphorylated and inactivated by MK2. These mice were strongly protected against pathological consequences of inflammation in several infectious and non-infectious disease models. We also described evidence that TTP accumulates in a phosphorylated and inactive form at sites of inflammation. These pieces of evidence suggest that anti-inflammatory effects could be exerted by increasing PP2A activity and driving the dephosphorylation and activation of TTP. Loss of PP2A activity is a common causative event in several types of cancer. PP2A-activating drugs (PADs) are considered very promising anti-tumour agents, and are generally well tolerated in vivo. We will investigate whether two such compounds exert anti-inflammatory effects via the dephosphorylation and activation of TTP. We propose a multidisciplinary approach using biochemical, molecular biological, bioinformatic and imaging methods to characterise effects of PADs upon macrophages, which are key mediators of inflammatory pathology. TTP mutant mice and cells derived from them will be used to rigorously test mechanisms of action of the drugs. As a further step towards translational development, we will also test anti-inflammatory effects of PADs in inflamed human synovial tissue ex vivo.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Mucosal Postcodes - Understanding T-Cell Trafficking in the Oral Mucosa
Funder: Medical Research Council

Abstract: Leukocytes must traffic precisely to the oral mucosa to mediate their protective effects, or disease-causing potential in health and disease. This proposal seeks to address the currently limited insights into the molecular mechanisms regulating homeostatic and antigen-specific immune cell migration to the oral mucosa. We hypothesise that chemokines, the main in vivo regulators of leukocyte migration, have a key role regulating the trafficking of T Cells, to the oral mucosa. Using our expertise in chemokine biology and established techniques for analysing lymphocytes from the mouse oral mucosa, we will isolate T Cells that have travelled to specific murine oral mucosal sites. The transcriptome of the isolated cells T Cells from resting and antigen challenged tissue will be analysed using RNASeq. This will offer us in-depth and unbiased analysis of the T Cell transcriptome and identify molecules essential to T Cell trafficking in oral mucosa. Corresponding chemokine ligands in associated tissue will be analysed using PCR-array-based techniques. We will investigate candidate molecule inhibition in murine models using knockout mice and pharmacological blockers. Expression of candidate molecules will be assessed in human oral mucosa and T Cells from this mucosa, in health and disease using PCR-array approaches. We propose saliva plays a key role in the homing of T Cells to oral mucosal tissue. This will be investigated by culturing T Cells with saliva and assessing their expression of the candidate molecules identified. The homing ability of saliva-treated T Cells in vivo will be assessed by adoptive transfer of the in vitro treated cells. These data will define the expression of chemokine receptors, and other molecules associated with T Cell homing to the oral cavity. Developing our understanding of oral leukocyte migration may lead to the identification of new therapeutic targets for multiple debilitating oral diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: CHARACTERISING ARAP3-MEDIATED INTEGRIN INACTIVATION IN THE NEUTROPHIL
Funder: Medical Research Council

Abstract: The proposed work explores the mechanism by which neutrophil integrins are inactivated. It builds upon our current work, which demonstrates that the GTPase activating protein (GAP) ARAP3 is required for neutrophil integrin inactivation, thereby promoting neutrophil recruitment to sites of inflammation. ARAP3 is a PI3-kinase and Rap regulated GAP for Arf6 and RhoA. Rap, RhoA and Arf6 are all known regulators of leukocyte integrins. The proposed work will address how signalling through Rap-ARAP3-Arf6/RhoA regulates integrin inactivation in the neutrophil. We will employ conditionally HoxB8-immortalized hematopoietic progenitors, which will be differentiated to neutrophils in vitro. Unlike primary neutrophils, HoxB8 progenitor cells are amenable to genetic modification. Arap3-/- HoxB8 neutrophils will be engineered to express physiological amounts of ARAP3, or of ARAP3 incorporating individual point mutations that uncouple it from activation by Rap or that render it ArfGAP and/or RhoGAP dead. In particular we will elucidate (i) any role of Rap-ARAP3-RhoA signalling in integrin inactivation and (ii) any role of the ARAP3-Arf6 axis in integrin trafficking. In addition, integrin-dependent effector functions including migration and transendothelial migration in vitro as well as neutrophil recruitment in an adoptive transfer model in vivo will be analysed. The proposed work will contribute to our understanding of the regulation of integrin inactivation, a poorly defined, though important facet of integrin regulation, which is already recognized as a therapeutic target. The proposed work may lay the foundations to the development of new therapeutic approaches targeting neutrophil integrin inactivation in chronic inflammation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Defining the roles and functional interdependencies of linear ubiquitin-related deubiquitinases and LUBAC in TNFR1 signalling
Funder: Medical Research Council

Abstract: Ubiquitination events are major regulators of immune receptor signalling. Tumour necrosis factor (TNF) is a crucial cytokine for the development of an effective immune response to infections. It is therefore important that TNF can act in the physiologically desired manner. However, in many diseases, including several auto-immune diseases but also in certain inflammatory-driven cancers, TNF signalling is perturbed. The main receptor for TNF is TNF receptor 1 (TNFR1). TNFR1 signalling is exquisitely regulated by ubiquitination and de-ubiquitination events. We and others have recently shown that LUBAC, the only known ubiquitin ligase specific for the generation of M1-linked ubiquitin chains (linear ubiquitin) is recruited to the TNFR1 signalling complex (TNFR1-SC) where it is required for optimal gene-activatory signalling whilst limiting TNF-induced cell death. Maintaining the balance between these two outcomes is crucial for both, TNF's role in immunity to infection and in preventing it from inducing auto-immunity. In this proposal, we aim to understand the role of LUBAC and LUBAC- or linear-ubiquitin-related deubiquitinases (DUBs) in the regulation of the different TNF signalling outputs, i.e. gene activation and cell death. In particular, we will study how LUBAC and linear ubiquitination regulate these three DUBs and how these DUBs regulate and affect linear ubiquitination in TNFR1 signalling and the various functional outputs of TNFR1 stimulation. We are also interested in studying the inter-dependencies of these DUBs in regulating LUBAC and linear ubiquitin. Defining the specific and inter-dependent roles of these factors shall reveal important aspects of the biology of TNF and will hopefully inform future therapeutic strategies which we hope will rely on specific targets that only interfere with certain, pathological aspects of TNFR1 signalling instead of systemically inhibiting TNF which is the current standard approach in the clinic.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of type 2 innate lymphoid cells in autoimmune islet infiltration and diabetes
Funder: Medical Research Council

Abstract: In recent years it has become apparent that innate lymphoid cells (ILC) can potently influence immune responses. They can directly elicit innate effector functions and can also shape the nature of adaptive immune responses. We have identified a population of type 2 ILC (ILC2) in the pancreas of mice developing autoimmune diabetes. We hypothesise that the presence of ILC2 at this site will influence the complex cellular cross-talk involved in pancreatic beta-cell destruction. In support of this, our preliminary data show that pancreatic ILC2 reprogramme their effector functions in response to islet-infiltrating T cells, and that ILC2 products can alter T cell production of diabetes-relevant cytokines. In this project we will dissect how the bi-directional interplay between local ILC2 and islet-specific T cells influences the emerging autoimmune response, using state-of-the-art tools to unpick the molecular and cellular pathways responsible. We will define precisely how ILC2 change following T cell invasion of the pancreatic islets and unravel the downstream consequences of this for the immune response. Stimulated by our preliminary transcriptional analysis, we will test whether ILC2 can sense dying beta-cells and examine ILC/neuronal cross-talk. Finally we will use new models to test the contribution of ILC2 to diabetes development, and establish whether circulating ILC populations are altered in type 1 diabetes patients. These experiments have direct implications for our understanding of disease pathogenesis in type 1 diabetes and will fill a significant knowledge gap in the field. Notably many of the T-cell directed immunotherapies being trialed in diabetes would also be predicted to affect ILC2, so establishing whether these cells promote or inhibit disease is of high importance. Insights gained from the project may also be of broader significance to autoimmune tissue infiltration in other disease settings.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Defining the role of CAR in lung homeostasis and response to inflammation
Funder: Medical Research Council

Abstract: Coxsackie virus and adenovirus receptor (CAR), is a member of the immunoglobulin (IgG) superfamily that functions as a cell-cell adhesion molecule through homophillic interaction in trans. CAR was originally identified as a primary receptor for Adenovirus cell binding. However, our work and that of others has demonstrated that CAR also has a key role in stabilising epithelial cell-cell adhesions as both an adherens and tight junction localized protein. As well as forming homodimers in trans, CAR has also been shown to bind in trans to other members of the JAM family proteins, including JAM-L that is expressed on the surface of leukocytes and this facilitates leukocyte transmigration and activation. Our recent studies have shown that CAR levels in epithelial cells control junctional protein dynamics and that this is in part regulated through phosphorylation of the CAR C-terminus at S290/T293 by PKC. Our unpublished data shows that conditional depletion of CAR from the epithelial cells in mouse lungs leads to a dramatic reduction in inflammatory responses to house dust mite. These findings place CAR as a key novel molecule in regulation of the interplay between epithelial and immune cells and thus of potential relevance in the development and progression of lung diseases such as asthma and COPD. Our present study will define how CAR elicits a pro-inflammatory effect in the lung and analyse the pathways and CAR-binding proteins that regulate this process. We will also define how CAR associates with itself across adjacent cell membranes, and determine whether alterations to the underlying matrix (as occurs in asthma) also leads to altered CAR function and disease exacerbation. Data arising from this study will provide new insight into CAR function in the epithelium and potentially enable the development of novel therapeutic approaches in future.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Defining and predicting the innate immune response to critical injury
Funder: Medical Research Council

Abstract: Samples: This research will use existing and future blood samples from critically injured patients (ISS 25+) collected at the Royal London Major Trauma Centre in the "hyper-acute window" (within 2 hours of injury). Samples are processed for plasma, protease-inhibited plasma, and leucocytes (as PAXGENE and buffy coat). 676 existing samples meeting study criteria are available. Methodologies: Aim 1: To understand the relationship between tissue damage and advserse clinical outcomes by; 1.1: Whole genome transcriptomic analysis using RNA sequencing on samples taken in the hyper-acute window 1.2: Measurement of objective intrinsic indicators of tissue damage and inflammation-associated cytokines/chemokines 1.3: Latent class mixed modelling machine learning approaches combined with support vector algorithms Methodologies: Aim 2: To gain mechanistic insight into differential hyper-acute immune responses to trauma, by; 2.1: Single cell immune cell profiling using mass spectrometry (cyTOF) on a prospective cohort 2.2: Measurement of plasma levels of IL-6, sIL-6R, and sgp130 in our new cohort 2.3: 10X Genomics single-cell-RNA-seq platform to generate single-cell transcriptomic data for 5-10K purified PBMCs to identify differentially regulated biomarkers and critical signaling modalities 2.4: Flow cytometry & functional analyses of leukocytes from healthy volunteers cultured with hyper-acute plasma from trauma patients who subsequently developed (or did not) MODS Exploitation of results: Key applications will be; A) The generation of new knowledge in understanding the human immune response to tissue damage and its relationship with MODS and immunosuppression; B) Hyper-acute biomarkers and stratification tools for prognostication, clinical trials and precision approaches to resuscitation; and C) novel targets for therapeutic repositioning and drug discovery to reduce the incidence and severity of organ dysfunction and infection in trauma patients.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM