Regulation of Pulmonary Neutrophils In Vivo: Direct Interrogation by Intravital Microscopy
Lead Research Organisation:
Imperial College London
Department Name: National Heart and Lung Institute
Abstract
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.
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.
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.
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.
Publications
McCormick B
(2019)
A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.
in Journal of immunology (Baltimore, Md. : 1950)
Ntala C
(2021)
Analysis of Prostate Cancer Tumor Microenvironment Identifies Reduced Stromal CD4 Effector T-cell Infiltration in Tumors with Pelvic Nodal Metastasis.
in European urology open science
Leslie J
(2022)
CXCR2 inhibition enables NASH-HCC immunotherapy
Leslie J
(2022)
CXCR2 inhibition enables NASH-HCC immunotherapy.
in Gut
Pillay J
(2020)
Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen
in Journal of Leukocyte Biology
Schmidt T
(2023)
eIF4A1-dependent mRNAs employ purine-rich 5'UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation.
in Nucleic acids research
Fercoq F
(2019)
IL-4 receptor dependent expansion of lung CD169+ macrophages in microfilaria-driven inflammation.
in PLoS neglected tropical diseases
Duarte D
(2018)
Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML.
in Cell stem cell
Secklehner J
(2017)
Intravital microscopy in historic and contemporary immunology.
in Immunology and cell biology
Kostelec, Pedro D.
(2015)
LEARNING TO DETECT AND TRACK CELLS FOR QUANTITATIVE ANALYSIS OF TIME-LAPSE MICROSCOPIC IMAGE SEQUENCES
in 2015 IEEE 12th International Symposium on Biomedical Imaging (ISBI)
Karadjian G
(2017)
Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung.
in PLoS neglected tropical diseases
Matchett K
(2023)
Multimodal decoding of human liver regeneration
Brown E
(2018)
Multiple membrane extrusion sites drive megakaryocyte migration into bone marrow blood vessels.
in Life science alliance
Patel DF
(2019)
Neutrophils restrain allergic airway inflammation by limiting ILC2 function and monocyte-dendritic cell antigen presentation.
in Science immunology
Puttur F
(2019)
Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans.
in Science immunology
Irshad S
(2017)
ROR?t+ Innate Lymphoid Cells Promote Lymph Node Metastasis of Breast Cancers.
in Cancer research
| Description | Imperial College PhD Scholarship |
| Amount | £96,650 (GBP) |
| Organisation | Imperial College London |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 09/2015 |
| 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 | 03/2016 |
| 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 | 05/2015 |
| 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 | 05/2016 |
| 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. |
| 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 |