Masters of research training programme in Respiratory Medicine and Infectious Disease

Type: Immunology,Translational, wet lab     

Disease areas: COVID19, chronic lung disease mechanisms 

The DNA and protein webs explosively released from neutrophils known as neutrophil extracellular traps (NETs) are implicated in the pathogenesis of a number of lung diseases including acute COVID-19 and bronchiectasis. NETs can provide a long-lived delivery system for harmful neutrophil proteases into the lung tissue and can impair neutrophil-mediated bacterial clearance. Resolution of inflammation involves degradation and removal of NETs by macrophages. However, mechanisms of NET removal are poorly characterised. In addition, it has been reported that macrophage interaction with NETs can alter key activities of these cells, potentially enhancing antimicrobial activity. Treatments that could enhance resolution of NET-mediated inflammation have potential to treat multiple acute and chronic diseases. Students will generate monocyte-derived macrophages (MDM) from peripheral blood from patients with lung disease and healthy controls. NETs will be generated from primary human neutrophils. The interaction between NETs and MDMs will be studied within NET degradation experiments, nuclease activity from stimulated and unstimulated MDMs will be measured and the effect of MDMs on experimentally-induced neutrophil extracellular traps, and vice versa, investigated in-vitro. Key techniques for which full training will be provided include primary cell isolation, tissue culture, time-lapse microscopy and image analysis, and immunoassays. 

Type: Microbiology, translational, wet lab. 

Disease areas: Antibiotic resistance, bronchiectasis. 

Antimicrobial resistance is a major threat to human health globally. It is not known why following exposure to antibiotics some organisms develop resistance and others do not. Doxycycline is a tetracycline antibiotic that is among the most frequently used for human infections, but resistance is also known to develop rapidly. You will work with a multidisciplinary team to investigate the effect of treatment with doxycycline for 28 days in patients with chronic lung infections to investigate the effect of antibiotic treatment on antimicrobial resistance. Bacteria from the airway will be cultured with testing for antimicrobial resistance. Isolated bacteria from before and after treatment will undergo whole genome sequencing to identify genetic changes associated with tetracycline resistance. This will be complemented with in-vitro experiments exposing bacteria to increasing concentrations of doxycycline to study bacterial adaptation. The aim of the project is to identify why some microorganisms readily develop resistance to doxycycline to develop personalised antibiotic strategies.

Type: Cell culture, translational, wet lab. 

Disease areas: Mucociliary clearance, Lung infections. 

Bacteria cause infections by damaging and binding to the epithelial barrier. Damage to the epithelium causes inflammation and impaired epithelial function which causes much of the manifestations of human disease. Understanding epithelial damage in the context of infection is important to identify new treatment targets and to study disease. This multidisciplinary project will examine experimentally damaged as well as “in vivo” epithelial damage to develop novel biomarkers based on microscopy/imaging, ELISA or other methods to identify and quantify epithelial damage in response to infection. Core work will involve culturing air liquid interface epithelial cultures, assessments of epithelial function, and experimental infections using bacteria or bacterial/host products to induce epithelial disruption. 

Type: Immunology,Translational, wet lab     

Disease areas: COVID19, chronic lung disease mechanisms 

Neutrophils are the key first responders in acute and chronic respiratory infections but currently there are no drugs which directly target neutrophils. Identifying new targets requires a deeper understanding of the neutrophils behaviour under inflammatory stress. In this project neutrophils isolated from healthy donors and patients with disease will provide neutrophils for parallel proteomic and transcriptomic profiling at rest and following stimulation with classical host derived (e.g C5a) and bacterial (LPS) stimuli. Circadian changes will also be probed by evaluating sampling from subjects in the morning and at night. This novel exploration will provide new insights into neutrophil biology leading to new drug targets. 

Type: Wet lab, microbiology, immunology, cell culture   

Disease area: bronchiectasis, cystic fibrosis, chronic infection

P. aeruginosa is an opportunistic pathogen that frequently infects individuals with cystic fibrosis and bronchiectasis. P. aeruginosa infection typically drives a Th1/Th17 neutrophil-mediated immune response; however, increasing evidence highlights a link between P. aeruginosa infection and T2 responses in those with chronic infection and/or bronchiectasis as a means to promote/prolong survival. Recent evidence from our lab also highlights P. aeruginosa as a potential contributor to the development of a clinically relevant endotype of disease termed ‘eosinophilic bronchiectasis’, which has thus far been associated with increased disease severity and a risk of exacerbation. In this project, students will investigate the inflammatory cascade elicited by respiratory epithelial cells and isolated peripheral blood mononuclear cells following exposure to P. aeruginosa and whether these responses are strain- or patient-specific. This project will comprise various experimental techniques, including cell and bacterial culture, isolating cells from peripheral blood, measurement of inflammatory mediators by ELISA/multiplex cytokine panel and flow cytometry, with the opportunity for analysis of bacterial sequencing data. 

Type: immunology, Translational, wet/dry lab     

Disease areas: COVID-19, acute and chronic lung infections 

Exogenous corticosteroids (dexamethasone) are standard of care for patients with COVID-19 and have been shown to cut mortality by up to 30% in acutely ill patients. Recent data suggests that patients with ongoing symptoms post-COVID19 have low levels of the endogenous steroid hormone cortisol. Therefore patients with covid experience a marked spike in steroid followed by relative steroid deficiency in cases of ongoing symptoms. We have recently shown profound change in the immune cell proteome at 1 month after COVID-19 infection. In this study, using clinical samples and in-vitro experiments treating immune cells with cortisol and other steroids, we will profile the changes in immune cell function induced by corticosteroid treatment with the aim of understanding novel targets for acute and “long covid”.

Type: immunology, Translational, wet/dry lab     

Disease areas: COPD, bronchiectasis, asthma 

Prior work in our lab has shown an increased risk of chest infections/exacerbations of chronic lung disease during days when the levels of air pollution increase, suggesting that air pollution contributes to susceptibility to lung infections. The reasons for this are unclear, since most chest infections are believed to be caused by bacteria or viruses. We hypothesise that exposure to air pollution increases susceptibility to bacterial exacerbations through impairment of clearance of bacteria from the lungs. In a clinical study you will measure individual exposure to air pollution through measurement of black carbon in blood, a validated marker of environmental air pollution exposure. This will be related to individual risk of chest infections using molecular analysis of the airway microbiome (including sequencing and data analysis). You will model interactions between air pollution in epithelial cell cultures by culturing cells with pollutants, followed by exposure to bacteria to study how this changes the release of antimicrobial peptides. This research aims to understand how potential treatments could protect people with lung disease from the effects of air pollution. 

Type: Immunology,translational, tissue culture, wet lab    

Disease areas: chronic lung disease mechanisms 

Treatments targeting inflammation, and particularly the use of biologics, have had a highly beneficial impact on outcomes for people with acute and chronic lung diseases. Blockade of the epithelial alarmin IL-33, released in response to damage, is a novel treatment strategy in development for people with asthma, COPD and other chronic lung conditions like bronchiectasis.

Animal studies suggest that IL-33 blockade reduces neutrophilic inflammation in the lungs but whether this is a direct or indirect effect is unknown. Understanding how IL-33 blockade can influence inflammatory responses in the lungs is important to determine the future role of IL-33-blocking monoclonal antibodies in disease. This project will investigate these mechanisms of action in multiple cell types, namely primary human neutrophils and primary airway epithelial cells obtained from patients with lung disease and control subjects. In addition to primary cell isolation and tissue culture, key laboratory techniques for which full training will be provide include flow cytometry, immunohistochemistry and microscopy, ELISA, functional assays, and analysis of large datasets.

Type: Immunology,translational, tissue culture, wet lab    

Disease areas: chronic lung disease mechanisms 

In acute and chronic respiratory diseases like COPD and acute respiratory distress syndrome, the dysfunctional activity of lung macrophages contributes to disease progression. Key dysfunctional activities include macrophage antimicrobial action, inflammatory activation and ability to maintain tissue homeostasis by activities such as the removal of apoptotic cells. However, the role of lung macrophages in bronchiectasis has not yet been explored. This project will utilise a macrophage cell line and primary human monocyte-derived macrophages generated from blood samples from people with chronic lung disease (COPD or bronchiectasis) and controls to develop a multiparametric flow cytometry-based assay for the measurement of macrophage functions. Key analyses include inflammasome activity, bacterial phagocytosis, macrophage activation, and reactive oxygen species production. The assay will then be used to investigate whether dysfunctional macrophage activity plays an important role in bronchiectasis. In addition, the project will explore the role of IL-1B and inflammasome activation overall in bronchiectasis using stored samples from the respiratory group biobank and data from previous sample analyses. Key techniques for which full training will be provided include primary cell isolation and culture, flow cytometry, live-cell imaging, ELISA, and analysis of large datasets.

Type: Immunology,translational, tissue culture, wet lab    

Disease areas: Chronic lung disease mechanisms 

Aberrant cell death and cellular senescence both have a role in chronic inflammatory lung diseases such as COPD to promote harmful disease processes. Analyses in our lab have recently suggested differences in individual patients’ rates of neutrophil apoptosis in those with the chronic lung disease bronchiectasis, in addition to high levels of senescence-associated proteins including IL-6 and IL-8 in the airways associated with more severe disease. Delayed neutrophil apoptotic cell death can increase the propensity for damaging neutrophil activity in the lungs and increased numbers of senescent cells have also been identified in chronic lung diseases and linked with “inflammaging”, the process whereby premature cell ageing drives inflammation. It is not known precisely if or how these processes play a role in bronchiectasis. 

This project will investigate the links between altered cell death mechanisms, cellular senescence, immune function and clinical parameters including disease severity. Using existing data from clinical translational studies, senescence-associated proteins measured in airway samples will be grouped and correlated with severity and other clinical parameters to investigate associations. Samples will be utilised from an ongoing clinical study enrolling people with bronchiectasis and matched controls to isolate peripheral blood neutrophils and measure classical apoptosis and NETotic cell death, levels of pro-survival factors, as well as other immune cell functions. Lung tissue biopsy samples from the same individuals will undergo immunohistochemical staining for markers of senescence to determine associations with inflammation and immune function.

Therapies targeting neutrophil proteases, which in particular can be explosively released when neutrophils undergo a form of cell death called NETosis, have recently been highly successful in bronchiectasis. In addition, therapies targeting aberrant cell senescence have been proposed for other chronic lung diseases, and may be useful in bronchiectasis if these mechanisms are found to play a role. 

Key techniques will include human peripheral blood neutrophil isolation, functional analyses including fluorescence microscopy and in-plate fluorescence assays, live cell imaging, analysis of tissue sections after histology staining, ELISA, and data analysis. Full training will be provided.

Type: Immunology,translational, tissue culture, wet lab    

Disease areas: Chronic lung disease mechanisms 

Changes in immune cell metabolism (i.e. immunometabolism) impact the function and antimicrobial capacity of these cells. Neutrophils are innate immune cells which can drive chronic lung disease including bronchiectasis, where they become overactivated and dysfunctional, contributing to airway damage and persistence of infecting microbes. In chronic obstructive pulmonary disease (COPD), altered neutrophil metabolism promotes defective microbial clearance and harmful neutrophil activation. 

Using transcriptomics, our lab recently identified differences in metabolic pathways in blood immune cells from people with bronchiectasis compared with controls. Therapies modifying cell metabolism are currently available and may be useful in chronic lung diseases to modulate dysfunctional immunometabolism and therefore alter immune cell function. This project will characterise neutrophil and peripheral blood mononuclear cell metabolism in bronchiectasis and matched control participants before and after treatment with inflammatory stimuli, alongside parallel investigations of cell functional capacity including cell survival and antimicrobial capacity. The aim of the project is to determine whether immunometabolism is altered in bronchiectasis and to understand the functional impact of any differences. Key techniques will include primary immune cell isolation, flow cytometry, live-cell imaging, seahorse metabolic flux analyser assays, an in-plate fluorescence assays. Training will be provided. 

Type: Immunology,translational, wet lab     

Disease areas: Chronic lung disease mechanisms 

The circadian clock controls core bodily functions, including sleep, hormone release and regulation of body temperature. This clock goes by the 24-hour day and is set by environmental factors such as light. The circadian clock exerts its effects via regulation of gene and protein expression by key transcription factors. Research has shown diurnal changes in neutrophil function and potentially harmful neutrophil protease release are regulated by the circadian clock genes and by the chemokine receptor CXCR2 rhythmically expressed on the neutrophil cell surface. Links have been identified between circadian rhythm and outcomes in patients hospitalised with acute respiratory infections, and in asthma the circadian clock is implicated in the worsening of symptoms overnight. 

Neutrophils have a critical, damaging role the chronic inflammatory lung disease bronchiectasis, associated with airway inflammation, destruction and infection, and recently therapies targeting neutrophil proteases have been shown to be highly successful. In chronic lung diseases, circadian rhythm may be dysregulated and could contribute to disease features such as inflammation and airway microbiome dysbiosis. 

Within the present project, blood samples from study participants with and without bronchiectasis will be collected, stabilised and stored and expression of key clock genes analysed in the morning and afternoon by qPCR. Using stabilised, stored peripheral blood immune cells, a multi-colour flow cytometry panel will be developed to perform immunophenotyping analyses to determine cell proportions and activation. In a participant subset, peripheral blood neutrophils will be isolated from healthy individuals in the morning and afternoon and will undergo untargeted proteomic and transcriptomic analysis to determine circadian influence on the whole cell profile and to define key downstream effects of circadian rhythm for further investigation. Where sample timing has previously been recorded, to evaluate circadian impact on airway inflammation, levels of key inflammatory markers will be measured in stored sputum samples from the BRIDGE European Bronchiectasis Registry biobank and stratified by time of day. This project aims to determine how the circadian clock impacts immune cell function in bronchiectasis and whether the clock is dysregulated, in order to inform future treatment strategies.

Key techniques include multi-colour flow cytometry, primary human immune cell isolation, qPCR, ELISA, and data analysis. Full training will be provided.