Pilot award reports
Visualising early life dendritic cell immune-regulation in lung inflammation
Early life is a period of heightened susceptibility to infections and risk of developing chronic inflammatory diseases. Vaccines are key to reducing the economic burden of paediatric infections and chronic diseases. Development of therapies that implement early life immunizations is vital for reducing deaths in neonates and children world-wide. Due to striking quantitative and qualitative differences in the early life environment, it is mandatory that an in-depth understanding of environmental factors regulating the early life immune system is needed for the development of effective early life vaccines. The early life immune system is characterized by unique immune cell developmental changes which are influenced by specific microbial exposures and extracellular matrix changes. After birth the immune system that normally develops in a relatively sterile foetal environment emerges into one filled with diverse environmental encounters. With this pilot award, I was able to interrogate the airway extracellular microbial and extracellular matrix environment in the first weeks of postnatal life and following allergic airways inflammation. I was able to generate an Imaging mass cytometry protocol that allowed me to capture cell-matrix associations and changes in immune cell (including dendritic cell subtypes) phenotype and activation status. My data has generated a resource to interrogate multiple biological interactions involving dendritic cells, and other immune cells while simultaneously tracking changes to the extracellular matrix density, composition, texture and geometry. These data will provide an important foundation to interrogate specific cell-matrix interaction and provide clues to functional regulation of Dendritic cells in an early life environment
Exploring Aspergillus fumigatus gene expression during co-culture with Pseudomonas aeruginosa
Chronic respiratory infections are the leading cause of mortality in patients with CF. The complex bacterial community within the lungs has been well established, however, fungal infections are being increasingly recognised as a factor in poor clinical outcomes. Aspergillus fumigatus (Af) is one of the most common fungal pathogens isolated from CF lungs yet in many cases it is unclear the role Af is playing in disease progression.
In a recent study with Dr Dominic Hughes, co-culture of the Af and the CF bacterial pathogen Pseudomonas aeruginosa (Pa) was found to be uncommon (1). However, sequencing data revealed Af in culture negative samples in the presence of Pa. To further explore these interactions this project explored gene expression of Af strains from CF patients in the presence of Pa.
Using the Oxford Nanopore MinIon I performed RNAseq analysis on Af strains grown alone and in the presence of Pa. This analysis revealed several differentially expressed genes in the presence of Pa. These genes were involved in several processes, importantly two key genes involved in growth and development were upregulated in the presence of Pa. These genes were StuA, a gene involved in developmental competence, and IdoC, a key gene in l-tryptophan metabolism. The upregulation of these genes suggest increased hyphal growth during co-infection. The increase in tryptophan metabolism may also be related to increases in fungal toxins (e.g. fumiquinazolines, gliotoxin, and fumitremorgins) which are downstream metabolites of the tryptophan pathway.
Surprisingly no significant difference in genes associated with Iron starvation were observed in this data. This is likely due to the nutrient rich media used for these experiments. Further work will involve the management of nutrients in this system to understand how fungal gene expression in the presences of Pa is changed under more clinically relevant conditions.
This award has also been invaluable in securing a lectureship at the University of Leicester where I will continue this work to understand fungal and bacterial interactions in respiratory disease. The preliminary data collected in this project will be a key in my application for a career development award which I plan to submit next year.
Engineered Pulmonary Artery Tissues
Current conventional techniques to culture smooth muscle cells (SMCs) in vitro do not reproduce their native environment and thus yield inaccurate results. This has substantial negative repercussions for the study of diseases involving SMCs, particularly cardiovascular diseases such as pulmonary arterial hypertension (PAH) which has a high mortality rate and for which there is no cure.
The NHLI Pilot Award has allowed me to develop “Engineered Pulmonary Artery Tissues” (EPATs) – a transformative method for culturing SMCs which is more physiologically relevant and has the potential to vastly improve the process for finding new and better treatments for PAH.
Findings From This Award
We have found that EPATs recapitulate both vasoconstriction and vasodilation in response to a range of drugs including KCl, endothelin-1, U46619, sildenafil and sodium nitroprusside – all of which are clinically relevant in the treatment of PAH
Our results also demonstrate that EPATs remain vasoactive for at least 3 weeks in culture, over which time multiple experiments can be performed. We have also ascertained the optimum number of cells needed per EPAT (1,000,000) in order to maintain contractility over this period
We performed a medium-throughput drug screening study in which we tested the vasodilatory impact of three established PAH therapies (bosentan, epoprostenol, selexipag), two emerging PAH therapies (imatinib, serelaxin) and one cardiovascular drug known to be ineffective in PAH (propranolol). Findings obtained in the clinic were reproduced in EPATs, which shows that they are an accurate mimic for studies of vasoactivity and for the first time we provided compelling evidence that both imatinib and serelaxin are potent vasodilators in the pulmonary vasculature
Interestingly, we also revealed significant variability in responses depending on the cell donor, which emphasises a need for greater personalisation when treating conditions such as PAH
The findings generated from this award has directly contributed to my award of an NHLI Research Fellowship for 2022-23, which I will use to further validate the translational potential of this technique and apply for a larger, externally funded fellowship to continue my research on EPATs
I would like to express my sincere gratitude to the foundation for this award and I hope the scheme will continue in the future so other early career researchers may benefit as I have.
Contributions by anion channels to platelet function
Platelet activation and blood clot formation is a major underlying cause of cardiovascular disease. For this reason, it is important to understand the processes that regulate blood clot formation to identify potential therapeutic targets. Platelet activation is a tightly regulated process and critically dependent upon the flux of charged ions such as calcium and chloride. Whilst the role of calcium is well understood, the mechanisms by which chloride, and other anions, contribute to these processes has received less attention. This is partially due to a lack of specific inhibitors that target anion channels. My Pilot Award application aimed to evaluate contributions by anion channels to platelet function using a combination of in vitro and in vivo techniques.
I was awarded £4,615.67 from an open competition of the NHLI pilot award scheme to generate preliminary data to be used for future grant and fellowship applications. I have 5.5 years of postdoc experience working on platelet-related projects and my aim is to establish my own research group.
I previously led a Wellcome Trust vacation scholarship that delivered a first-author publication, which demonstrated a role for platelet anion channels in regulating the rate of platelet activation (Taylor, KA et al., 2017). The underlying signalling pathways that governed this process remained unclear. Under this award the underlying anion channel-dependent signalling pathways were investigated using biochemical approaches. Whilst these experiments identified some potential candidate signalling proteins, experiments were prematurely ended due to the COVID-19 shutdown.
Anion channel blockers used in this study included two FDA approved drugs, which are well tolerated in humans. The effect of these drugs on thrombus formation was therefore studied in vivo using a refined mouse model of pulmonary thromboembolism. This technique was developed by the Emerson group (Cardio-respiratory Interface Section; NHLI) with funding from the National Institute for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). These studies highlight an inhibitory effect of upon thrombus formation, which indicate a physiological role for the target channels.
A major advantage of this Pilot Award was the opportunity to develop my independent collaborations, which will prove important for future grant applications. These collaborations were necessary to facilitate studies into platelet function beyond thrombosis and haemostasis. Given that circulating platelets contact the endothelium (blood vessel lining), it will be important to assess roles for platelet anion channels in mediating these interactions. For this purpose, I established collaborations with Dr Beata Wojciak-Stothard (Vascular biology; NHLI) and Dr Tom McKinnon (Haematology; Imperial College) to develop in vitro platelet-endothelial interaction assays. Whilst, some preliminary experiments were performed, this section of work was severely impacted by the COVID-19 shutdown.
I was made redundant in September 2020 due to a lack of research funds. I have since started a senior postdoctoral position at the University of Reading. I have retained an association with Imperial through an honorary research associate contract that will enable me to continue my collaborative research with Drs Wojciak-Stothard and McKinnon. Data from these collaborations will contribute to future grant and fellowship applications, enabling me to establish my independent research career.
Despite the COVID-19-associated setbacks, this pilot award was a valuable opportunity to generate exciting preliminary data that will assist me in the next stage of my career. In an increasingly challenging research funding environment, it is critical that such schemes exist to support early career researchers. Having served on the postdoc and fellow committee I can attest to the value of writing and scoring grant proposals submitted under this pilot award scheme, and I hope it will continue to support early career researchers within NHLI.
Deciphering the role of epigenetics of airway macrophages (AMs) in Idiopathic pulmonary fibrosis
I was awarded the NHLI Pilot Funding in January 2020 for a project looking to decipher the role of epigenetics of airway macrophages (AMs) in Idiopathic pulmonary fibrosis (IPF). Specifically, I profiled DNA methylation of AMs enriched from the lungs of IPF and healthy control donors and identified regions exhibiting differentially methylation. Importantly, these regions encompassed a number of genes implicated in metabolic processes which the Byrne lab had previously identified to be altered in IPF. These findings help to establish a link between epigenetics and phenotypic characteristics of AMs and their role in IPF pathogenesis.
Receiving the NHLI Pilot Funding has provided a tremendous boost to my career. Firstly, it allowed me to set up a cross institution collaboration involving UCL and QMUL to run and analyse the DNA methylation profiling experiments. This collaboration was extremely productive and has provided a foundation to pursue further studies of epigenetics in IPF. Secondly, the outcomes of the project have been submitted to the American Journal of Respiratory and Critical Care Medicine and received favourable reviews. As of this report, we are awaiting the response of reviewers and look forward to the works acceptance and publication in one of the most preeminent respiratory journals. Finally, the work conducted as part of the NHLI Pilot Funding contributed to my career by allowing me to obtain my current position as precision medicine scientist at BenevolentAI, a leading artificial intelligence and drug discovery company.
I wish to thank the NHLI foundation for providing me the opportunity to further my career and would hope with continued support of the Pilot Funding scheme, other scientists at NHLI will benefit as much as I have.
Understanding the mechanisms of increases neutrophil degranulation by hypoxia
Aims of the project supported by my NHLI Foundation Pilot Award were:
- What is the role of HIF in hypoxia augmented degranulation?
- Can hypoxic neutrophil-driven endothelial dysfunction be prevented by inhibition of PI3Kγ, my novel protein targets and/or HIF?
Outcomes and Impact:
I am very grateful to have received this NHLI Foundation Pilot Award which has enabled me to 1) establish an entirely new neutrophil-endothelial cell perfusion flow system housed within a hypoxic chamber which is unique to the NHLI, and 2) generate novel data to progress our understanding of the mechanism by which hypoxia increases neutrophil degranulation.
Aim 1: My original Pilot Award application requested funding to collaborate with Professor Johnson at the Karolinska Institute to access his HIF1α-knockout mice. Unfortunately, due to the impact of the Covid-19 pandemic on travel and their laboratory breeding programmes, I was unable to access the mice for these experiments. However, I took the opportunity to form a new collaboration with Professor Walmsley in Edinburgh and was thus able to access mice with a neutrophil-specific HIF1α deletion in order to examine the impact of HIF on neutrophil degranulation. Purchase of a murine neutrophil cell isolation kit funded by my NHLI Foundation Pilot Award allowed me to isolate neutrophils from wildtype and HIF1α-knockout mice. These experiments demonstrated for the first time that the hypoxic augmentation of neutrophil degranulation is HIF1α-dependent, and thus HIF1α may be an important target that could be exploited to prevent neutrophil-mediated cellular injury. These novel data will be presented at the HypoxEU conference in Dublin, September 2022, and will form part of my upcoming Fellowship application. Please note that these data are currently unpublished and remain confidential within the NHLI.
Aim 2: In collaboration with Dr Andrew Cowburn, NHLI, I have established a highly sensitive neutrophil-endothelial cell perfusion flow system. The flow system comprises endothelial cells in channel microslides which are perfused with isolated neutrophils. Neutrophil rolling,adherence and transmigration can be measured using fluorescence microscopy and time lapse video capture. My NHLI Foundation Pilot Award was used to fund a high specification video camera and software, allowing high speed video capture assessment of neutrophil recruitment. The video camera is fundamental to the ability of the perfusion flow system to generate reliable, reproducible and, importantly, quantitative data, as well as generating high quality images for use in presentations. The perfusion flow system is housed within a hypoxic chamber and this set up is unique to the NHLI. To establish this novel system, I have used the video camera to generate quantitative data in order to optimise thesetup in hypoxia, including assessing optimum: endothelial seeding density, neutrophil perfusion concentration, fluorescence staining protocol for neutrophils, degree of hypoxia (% oxygen concentration), timing of hypoxic exposure for endothelial cells and neutrophils, and pre-treatment of endothelial cells with priming agents and/or treatment with neutrophil supernatants. I have now generated preliminary data suggesting that hypoxia increases the ability of neutrophil supernatants to promote neutrophil-endothelial adhesion. My next step is to investigate whether inhibition of HIF or other signalling pathways can mitigate this proadhesive effect of hypoxia. My data, already presented at the Vascular Science Work in Progress meeting, will be used to support my upcoming Fellowship application. Furthermore, the setup can be expanded to accommodate many other cell types and has already been used collaboratively by other members of the Department for alternate adhesion assays.
Hypothesis and Aims
Hypothesis: Aspergillus fumigatus conidia do not attach efficiently to the surface of epithelial cells from CF donors compared with healthy, facilitating increased germination into hyphal form, and a greater inflammatory response.
- Train with Dr Claire Smith’s group (UCL GOS Institute of Child Health, London) to learn methodologies for growing ALI culture airway epithelial cells from CF patients.
- Use the ALI culture cells from healthy and CF donors to investigate Aspergillus fumigatus persistence on CF epithelial cells and the resulting inflammatory response.
Outcomes and Impact
The pilot award allowed me to train with scientists from Dr Smith’s group and optimise the protocols for growing primary nasal bronchial epithelial cells in the presence of inactivated mouse fibroblasts. This allows growth of larger numbers of cells for differentiation to air-liquid interface (ALI) culture. The award also allowed me to do the pilot work to investigate Aspergillus fumigatus (Af) growth on the surface of epithelial cells expressing either WT or Phe508del CFTR. Using the immortalised CFBE410- cells I was able to show evidence of increased growth of Af cells on cells expressing Phe508del CFTR. This data, along with the training in the cell culture was used in my successful application for the European Cystic Fibrosis Society and CF Europe Post-Doctoral Research Fellowship in 2020. Due to the COVID-19 pandemic the collection of nasal brushings for the project was not possible, therefore, a portion of the award was used to purchase primary bronchial epithelial cells from the McGill University primary cell biobank. I was able to get cells from six healthy donors and six donors with CF. Using the techniques from Dr Smith’s group I grew and froze down enough epithelial cells to produce over 200 transwells per donor. A small portion of the cells were used for this project, but the majority are in cryostorage and available for other members of Prof. Davies group for future projects. The ready grown transwells are very expensive to purchase, so the ability to produce large numbers of them from the stocks I produced and from future nasal brushings has great potential impact for the group. Using these ALI culture cells I was also able to start optimisation of Af infection on the surface of epithelial cells from healthy donors and those with CF and complete initial imaging of infected cells.
- Trained with Dr Smith’s group at UCL GOS Institute of Child Health, and made protocols available to Prof Davies group.
- Completed pilot work on immortalised cells, leading to successful application for the European Cystic Fibrosis Society and CF Europe Post-Doctoral Research Fellowship in 2020.
- Purchased bronchial epithelial cells from McGill university, using techniques learnt from Dr Smith to produce stocks for members of Prof. Davies group to grow large numbers of ALI culture cells for future projects.
- Optimised Af infection on the surface of ALI culture cells.
This grant enabled me to form a new collaboration with Prof Declan O’Regan and his team at the Robert Steiner MRI unit at the MRC LMS and Prof Oliver Rider and Dr Laci Valkovic at University of Oxford. We installed the cutting-edge 3-dimensional chemical shift imaging sequence for cardiac 31-phosphorus magnetic resonance spectroscopy on the 3T Prisma scanner at MRC LMS. The Oxford team trained us how to acquire and analyse the spectroscopy data. We scanned 8 healthy volunteers with improving interscan reproducibility. We also scanned 8 patients with recovered dilated cardiomyopathy who took part in the TRED-HF study. Early data suggest that patients with recovered dilated cardiomyopathy have reduced myocardial PCr:ATP compared to healthy volunteers. However, this requires confirmation in larger numbers of patients.
These data were also used to support the use of the sequence in my BHF Intermediate Clinical Research Fellowship. The randomised trial using spectroscopy has recently started. The pilot grant has allowed the team to receive adequate training and support in using the sequence prior to the start of the study. I am also preparing another Clinical Research Training Fellowship application which will include a larger, powered analysis comparing myocardial energetics in patients with recovered DCM and matched healthy controls.
Neutrophils possess an armamentarium of toxic products that are designed to destroy invading pathogens. However, in some respiratory diseases, despite the presence of significant neutrophil infiltration in the airways, chronic or persistent bacterial infection exists. Furthermore, there is excessive spillage of these neutrophil toxic products into the extracellular environment that inadvertently causes damage to the lung tissue and ultimately compromises lung function. This is particularly evident in cystic fibrosis (CF) patients where these patients display persistent airway infection with the gram-negative bacterium, Pseudomonas aeruginosa despite significant neutrophilic inflammation. It is important to understand how Pseudomonas adapts to withstand the antibacterial advances of the neutrophil. Such findings could ultimately aid in the development of therapeutic strategies to enhance neutrophil-mediated clearance of Pseudomonas from the CF airway and limit excessive neutrophil-mediated tissue damage.
With the NHLI Pilot award, I collaborated with Professor Jane Davies in the Strategic Centre for Pseudomonas Research to screen the effects of Pseudomonas that had been isolated from CF patients with acute and chronic infection on neutrophil antibacterial activity. It is important to consider that neutrophils are terminally differentiated and short-lived cells that are difficult to culture. Therefore, neutrophils must be assayed on the same day of isolation. This can restrain the ability to investigate neutrophil antibacterial activity, especially when assessing multiple bacterial isolates. Maximising the expertise, equipment and facilities within the NHLI and Imperial College London, I was able to develop unique high-throughput screening methods to assess the production of antibacterial neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) by neutrophils when cultured with bacteria.
With the help of these assays, I identified that Pseudomonas isolates from different infection timepoints of infection greatly differed in their capacity to induce NET formation and ROS production from neutrophils. Furthermore, I screened a series of genetically-modified Pseudomonas mutants lacking specific proteins which allowed me to dissect which Pseudomonas proteins/toxins disrupt or induce neutrophil killing functions. I identified multiple Pseudomonas proteins that differentially affect the neutrophil’s ability to form NETs and generate ROS during infection. Such a preliminary screen has generated a large quantity of novel data that has allowed me to formulate new research questions that will form the basis of future fellowship and grant applications.
In all, the NHLI pilot award has greatly allowed me to develop as an independent researcher. I was able to execute my own ideas and experimental plans in collaboration with leading scientists in the microbiology field. I performed research in a new lab setting and learned a range of new microbiology techniques which has allowed me to set up a new multidisciplinary programme of research that I wish to pursue in the future. The Pilot Award also gave me the freedom to develop high-throughput neutrophil screening tools that could be used for multiple purposes such as screening other bacterial species or drug screening. In addition to current collaborations, the preliminary data has allowed me to approach further collaborators and potential sponsors for fellowship or grant applications.
In spring 2019 the first round of NHLI Pilot Awards were advertised. As an early career researcher it can be very hard to find funding for even small projects, so I was very excited at the prospect of having the chance to apply. At the time of applying I had three epidemiological asthma studies that I had put forward as MSc projects. I realised that I could potentially use the award money to attain the data needed for them, such that I could be named primary supervisor, and the last author on any publications arising. This would not only help me develop my supervising skills but importantly give me the chance to show that I am leading on my own research ideas. This was particularly significant as I would shortly be applying for fellowships. At the time I specifically wanted to apply for a NIHR fellowship which is suited to my area of research, clinical epidemiology. A few months earlier at the NIHR Academy Day at Imperial College London they emphasised to us how important it was to have personal funding /grants on the application form, however big or small. The NHLI Pilot award would therefore considerably increase my chances of fellowship success by allowing me to evidence my independence and my ability to obtain competitive funding.
I was lucky enough to be successful in obtaining a NHLI Pilot award and the money was used to obtain the data required for the MSc projects. The MSc students all successfully completed their projects with abstracts presented at a national conference, which were well received, and for which I was last author. The manuscripts are being written up, again with the students as primary authors and myself as last author. I was declared as highly competitive in my NIHR application, and the reviewers specifically commented on my awards, including my NHLI pilot award. I am very grateful to the NHLI Foundation for the opportunity the Pilot award provided.