Regulation of Pulmonary Neutrophils In Vivo: Direct Interrogation by Intravital Microscopy

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute


Neutrophils are the most numerous white blood cells in the blood. They are critical in fighting disease causing microbes (bacteria, etc.). The blood vessels of the lung are extensive, very narrow and uniquely composed in order to allow the oxygen necessary for us to live into the blood and the carbon dioxide generated by our muscles and other organs to escape. When we breathe, microbes can come into close contact with the cells of the lung, therefore white blood cell function is critical in protecting the lung from infection. However, a lot of the processes that make neutrophils so good at fighting disease can be dangerous if not properly controlled. Neutrophils have been implicated in several 'inflammatory' lung diseases including acute respiratory distress syndrome caused by lung infections or injury, and severe asthma.
The way that neutrophils act can be regulated by several factors. Blood cells are produced in the bone marrow, and what stimulates their release into the blood can have marked effects on their function. Additionally, the cells that line blood vessels and the other white blood cells encountered can affect neutrophil function. New microscope technology allows us for the first time to look at white blood function in real lung blood vessels. This proposal investigates how neutrophils are regulated by building and combining new technologies to allow us to directly observe neutrophils in lung blood vessels and using these techniques to better understand the behaviour of neutrophils in healthy and diseased lungs.
The study aims to uncover fundamental aspects of neutrophil function, both beneficial in defence against microbes, and dangerous in terms of lung disease.

Technical Summary

Neutrophils are critical early mediators of inflammation involved in protective immunity to pathogens and implicated in the pathology of several lung diseases including acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and severe asthma. The microvasculature of the lung is unique in terms of how extensive and small the capillaries are and in particular with respect to their proximity to an external mucosa. The vascular endothelium in alveolar capillaries shares a basement membrane with the epithelium to facilitate gas transport. As such, tissue cells and cells within the capillaries are potentially under assault from the external environment. An understanding of the molecular mechanisms underlying inflammation in the lung is critical, since inflammation left unchecked compromises lung function.
This project develops and uses cutting-edge imaging techniques to directly interrogate how neutrophils are regulated in the lung microvasculature in situ to test the hypothesis that: Neutrophil regulation in lung homeostasis and inflammation is dependent upon bone marrow release, the lung microenvironment and their interaction with other leukocytes in pulmonary capillaries. Experiments will directly address these important influences in their physiological context.

Planned Impact

The findings of this study with make impact at several different levels:
Basic understanding of leukocyte function is critical to be able to build on to understand the pathogenesis of lung disease. These studies will provide fundamental understanding of how neutrophils are regulated in the blood vessels of the lung. This award will allow me to break new ground and provide the foundation of my own research programme, future publications and grant applications. This will be of great benefit my own future work to address more complicated disease pathogenesis, and also that of others in terms of generation of new knowledge and also the training of new scientists.
Intravital microscopy of leukocytes in the mouse lung is still a cutting-edge technology. To my knowledge, mine is the only UK lab where it is currently established. Its continued development and use to address the hypotheses here is a clear example of the development and utilisation of new and innovative methodologies, equipment, techniques and technologies. These skills will be shared with other researchers nationally and internationally. It will directly benefit students and post-doctoral trainees coming to the lab giving providing them with training in technology that has a great deal of potential across a number of disciplines.
Neutrophils have been implicated in the pathology of several serious and debilitating lung diseases, fundamental understanding of how neutrophils contribute to these diseases addresses globally important sources of mortality and morbidity.
I will also use the findings of the research to continue to contribute to increasing public awareness and understanding of science by taking part or continuing to take part in the Imperial Festival public engagement forum, the Pint of Science festival and continuing to co-organise Imperial CREST academy.


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Description Imperial College PhD Scholarship
Amount £96,650 (GBP)
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 03/2019
Description Keystone Symposia Future of Science Fund Travel Scholarship to Judith Secklehner (PhD student in my group)
Amount $1,200 (USD)
Organisation Keystone Symposia on Molecular and Cellular Biology 
Sector Charity/Non Profit
Country United States
Start 04/2016 
End 04/2016
Description NHLI Independent Research Fellowship to Katia De Filippo (Post-doc in my group)
Amount £31,440 (GBP)
Organisation Imperial College London 
Department National Heart & Lung Institute (NHLI)
Sector Academic/University
Country United Kingdom
Start 06/2015 
End 06/2016
Description Wellcome Trust Career Re-entry Fellowship to Katia De Filippo
Amount £624,438 (GBP)
Funding ID 201356/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 06/2020
Title Lung microscopy and analysis pipeline 
Description We have developed (in collaboration with M. Krummel, see collaborations) tools to determine and measure the localisation of immune cells in the lung, ex vivo (fixed and live lung slices) and in vivo (lung intravital microscopy). We have also developed new image segmentation tools (see publication). 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Publication in computer science journal (see publications). Several manuscripts in preparation for immunology journals. 
Title Mouse neutrophil maturity analysis in homeostasis, acute-inflammation and cancer - Bulk RNAseq data from sorted neutrophil populations 
Description Purpose: Identification of neutrophils at different stages of maturation and their phenotypic and functional differences remain unknown. The goal of this study was to confirm maturation status of Ly6GIntermediate, Ly6GHigh and Ly6G+ neutrophils, the consistency of this means of identication in disease, and identify functional differences. Methods: Ly6G+, Ly6GIntermediate and Ly6GHigh neutrophil populations were isolated by FACS into individual cell populations from PBS controls, acute-LPS inflammation and pancreatic adenocarcinoma (PDAC) bearing mouse tissues (metastatic KPC, non-metastatic KPflC and WT controls). RNA-Sequencing was performed on bulk isolated populations Results: We identified Ly6GIntermediate as bona-fide immature neutrophils and analysis of Ly6G expression level as a consistently accurate marker of identifying neutrophil maturity. Gene expression was influenced by maturation stage, tissue of isolation, developmental niche and pathology. Function associated expression differences were maintained between Ly6GIntermediate and Ly6GHigh neutrophils throughout tissues and pathology. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Dataset re-used in a comparison made in Leslie, Mackey, Jamieson et al. Gut 2022 
Description Intravital Microscopy of the Lung to Investigate Neutrophil Regulation 
Organisation University of California, San Francisco
Department Department of Pathology
Country United States 
Sector Academic/University 
PI Contribution Novel biological data, feedback on methodology
Collaborator Contribution Know-how and imaging adapter
Impact Have shared know-how and data, preparing a manuscript for submission
Start Year 2015
Description NK cell: Neutrophil Interactions 
Organisation Imperial College London
Department National Heart & Lung Institute (NHLI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Conceptual design of study and experiments, collection of data.
Collaborator Contribution Collaboration with Nadia Guerra, Transgenic mice, help, advice and know-how
Impact Abstracts / posters at Keystone Myeloid Cell Meeting (Ireland 2016) and British Society for Immunology (UK 2016)
Start Year 2015
Description Project to develop software for accurate identification of immune cells in intravital microscopy data 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of biomedical rational, data and evaluation, student supervision
Collaborator Contribution Software development and evaluation, student supervision
Impact Multidisciplinary, Biomedical Science and Computing. Kostelec PD, Carlin LM, Glocker B, 2015, Learning to detect and track cells for quantitative analysis of time-lapse microscopic image sequences, Pages: 1544-1547, ISSN: 1945-7928 Biomedical Imaging (ISBI), 2015 IEEE 12th International Symposium on DOI: 10.1109/ISBI.2015.7164172
Start Year 2014