How neutrophils detect and resolve infection and how this deteriorates with age

Lead Research Organisation: Babraham Institute
Department Name: UNLISTED

Abstract

Neutrophils play a key role in maintaining life long health and wellbeing. Neutrophils play a central role in maintaining human health. Patients with reduced neutrophil function die early and have severe symptoms following infection by minor pathogens. Neutrophils are adapted to contribute to the normal cycle of events that resolves infections. They migrate towards pro-inflammatory mediators released at sites of infection, they kill pathogens by phagocytosis, local production of reactive oxygen species (ROS) and release of proteases and they recruit further immune cells by release of pro-inflammatory mediators. In inflamed tissue neutrophils can be remodelled (“primed” to augment their functional responses) by the presence of cytokines like GM-CSF and TNFá which also delays their apoptotic death and thus increases their pathogen-killing capability. During recovery the levels of local cytokines return towards normal and neutrophils are lost by apoptosis. The molecules coordinating these activities are not fully understood although some key players have been clarified. GPCRs on neutrophils are key sensors of inflammation/infection and guide chemotaxis and stimulate ROS formation. A wide range of adhesive, pattern-recognition and antibody receptors allow neutrophils to generate some of the force needed for movement, stimulate ROS formation and allow pathogens to be recognised and phagocytosed. In past work we have found that class I PI3Ks heterodimers of catalytic and regulatory subunits that make the signalling lipid PIP3, and a number of RacGEFs (PRex1, Vav1 and Vav3; activators of the Rac-family of small GTPases, in neutrophils Rac1, Rac2, RhoG) are critical links between all of these receptors and physiological responses in isolated neutrophils and mouse models. However, the mechanisms that enable PIP3 and Rac signals to simultaneously or serially coordinate such complex behaviours remain unclear and depend on studying the different stages in the neutrophil response-to-infection cycle, defining molecular specificities and acquiring single cell and spatio-temporally-specific data. Our long-term aim is to understand, at a systems and molecular level, how class I PI3Ks and Rac GTPases coordinate neutrophil’s roles in host defence.

Publications

10 25 50

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Hornigold K, Tsonou E, Pantarelli C, Welch HC (2018) Encyclopedia of Signaling Molecules

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Mujalli A (2018) Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation. in Biochimica et biophysica acta. Molecular and cell biology of lipids

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O'Donnell VB (2018) Phospholipid signaling in innate immune cells. in The Journal of clinical investigation

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Pantarelli C (2018) Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment. in European journal of clinical investigation

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Pitchford S (2017) Platelets in neutrophil recruitment to sites of inflammation. in Current opinion in hematology

 
Description 1) We have identified ligand (hormone) specific loss of priming of mouse neutrophils with age. This has important implications for understanding of the age-related immuno-deficiency that is a major health problem for the elderly. 2) We have identified molecules that are are unexpectedly involved in protection against serious lung infections in vivo. 3) We have clarified the molecular mechanisms involved in PI3Kgamma signalling in mouse neutrophils and hence how PI3Kgamma plays a central role in pro-inflammatory signalling. 4) We have established the identity of signals in white blood cells that are important in restraining the immune system and maybe important in immune recognition and attack of tumours. 5) We have established proteomic methods needed to identify the proteins that organise PI3K signalling in neutrophils in health and how they may change with age. 6) We have identified the key role SOS-family RasGEFs play in regulating functional responses in primed and hence disease relevant neutrophils. Importantly, in unprimed cells the RasGEF, RasGRP4, is the dominant regulator of similar responses; offering a route to selective inhibition of functional responses in primed but not unprimed neutrophils.
Exploitation Route The results form part of the plans we laid out in our ISPG and will continue to be developed as described. The results are already impacting decision making and thinking in the pharmaceutical sector. Through our consulting work we have supported strategies aiming to inhibit PI3K signalling as cancer treatments by improving immune attack on the tumour cells specifically. We have also provided key data, measuring PIP3, to companies creating drugs to treat COPD. We have developed a substantial collaboration with AZ/Medimmune in the area of resistance to lung infection and have submitted a grant that if funded by RCUK will also be part funded by our industrial collaborators.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We have advised companies about the potential benefits of inhibiting PI3K signalling for improved immune detection and attack on tumours and this has provided part of the narrative that has led to PI3K inhibitors entering phase 2 trials in the MD Anderson (US) during the last 12months . We have helped develop assays and generate key data that has been used in a major company aiming to treat COPD by reducing immune activation via PI3K signalling in chronic, local inflammation. We have submitted a grant to a UK Research council in collaboration with AZ/Medimmune, and part funded by our commercial partners, to understand signalling controlling resistance to infection in the lung using mouse models.
First Year Of Impact 2014
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title Assay of PI(3,4)P2 in cells/tissues 
Description We developed a novel approach to quantify PI(3,4)P2 in biological extracts using mass spec based approaches. This knowledge has been transferred to other lipidomics facilities 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Used to assay the impact of tool compounds developed by PHARMA as activators of SHIP phosphatases that might be useful anti-inflammatories or anti-cancer drugs. 
 
Title Mice expressing Avi-tagged PI3K subunits and BirA 
Description Mice expressing endogenous C-terminally avi-tagged p85s (alpha and beta) and p110s (alpha, beta and delta) and mammalised BirA (from the ROSA26 locus) were generated. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? Yes  
Impact The tools have enabled the first studies of the underlying basis of selective interactions between PI3K subunits and with receptor RTKS in vivo. The molecular tools and mice have been transferred to other labs. These events have supported rational approaches to inhibition of PI3Ks as a therapeutic approach in inflammation and cancer. 
 
Title Mouse models with manipulated PLEKHS1 
Description Mice expressing endogenous avi-tagged PLEKHS1 were created using CRISPR/cas9 mediated targeting in zygotes. Mice in which the PLEKHS1 locus was conditionally manipulated were created using a "knock-out first" allele created by the EUCOMM consortium pipeline. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact The mice are being characterised. 
 
Title Novel mouse models with genetically manipulated PI3K gamma function. 
Description As we planned in the grant we have successfully created and vaildated a set of novel GM mouse lines in which we have knocked-in mutations into the endogenous PI3Kgamma regulatory (p101) and catalytic (p110g) subunits; point mutations that we have shown render them insensitive to G-protein beta gama subunits. We have also generated the first double regulatory subunit knock-out line (p84-/- x p101-/-; this was a non-trivial undertaking as the genes neighbour one another and hence inter breeding the single KO strains is extremyl unlikely to yield homozygous double KOs) . These mice have allowed us to determine the physiological roles of Gbeta gamma subunit binding to PI3Kgamma as distinct to regulation by for example Ras proteins. Hence in total we have created 4 novel lines (plus control strains) ; a p101-point mutant knock-in, p110g-point mutant knock-in, and strains expressing both mutations or lacking (KO) both p84 and p101 regulatory subunits. These unique resources form the basis of a large paper we are in the final stages of preparing, 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2016 
Provided To Others? No  
Impact It has enabled us to under take the first full dissection of the roles of Gbeta gamma subunits in regulation of PI3Kgamma in vivo and to establish that Gbeta gamma binding to p101 alone is physiologically important. We are finalising experiments and assembling a major paper with these results at the moment. 
 
Description Alison Condliffe 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution In vitro work with human neutrophils, measurements of phosphoinositides.
Collaborator Contribution Human patient samples.
Impact Publications fully reported
 
Description Chiesi 
Organisation Chiesi
Country United Kingdom 
Sector Private 
PI Contribution Measurements of phosphoinositides
Collaborator Contribution Development of PI3K inhibitors towards clinical trials
Impact Multi-disciplinary. We provide lipidomics expertise , Chiesi provide med chem and models
Start Year 2016
 
Description Doreen Cantrell 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurements of phosphoinositides
Collaborator Contribution Samples of lymphocytes
Impact Publications that are fully reported
Start Year 2014
 
Description GSK/Gus 
Organisation GlaxoSmithKline (GSK)
Department Respiratory Biology GSK
Country United Kingdom 
Sector Private 
PI Contribution Research into the physiological roles of Pi3kgamma in inflammatory signalling in neutrophils
Collaborator Contribution Knowledge of commercially important directions and implications of our experiments
Impact In the first year of the study.
Start Year 2011
 
Description I. Hers 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurements of phosphoinositides, supply of PI3K-GM-mice models.
Collaborator Contribution Platelet measurements
Impact Publications
Start Year 2016
 
Description Klaus Okkenhaug 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided mouse lines expressing avi-tagged pi3kdelta and birA for proteomic studies of lymphocytes we have supported proteomic analysis and pip3 measurements
Collaborator Contribution Advice over lymphocyte signalling
Impact Several co-authored papers with a further paper recently submitted.
Start Year 2010
 
Description Lindsay Hall 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Discussion/advice about possible roles of inhibitory receptors on immune cells in gut
Collaborator Contribution Advice about gut immune biology
Impact None yet
Start Year 2011
 
Description Orion Weiner 
Organisation University of California, San Francisco
Department Department of Biochemistry and Biophysics
Country United States 
Sector Academic/University 
PI Contribution Analysis of phosphoinositides, advice about neutrophils, reagents.
Collaborator Contribution Samples, reagents, technological advice.
Impact Publications fully reported.
 
Description Rob Kay 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Measurements of phopshoinositides
Collaborator Contribution Dictyostelium samples and reagents
Impact Publications that are reported in full
 
Description Role of SOS1/2 in neutrophil priming 
Organisation IEEC-CSIC
Country Spain 
Sector Public 
PI Contribution We provided the intellectual input and performed all the experiments
Collaborator Contribution They provided the SOS1/2 conditional KO mice and advice on using them
Impact Publication: PMID: 30720883
Start Year 2015
 
Description Simon Pitchford 
Organisation King's College London
Department Department of Pharmacology & Therapeutics
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration on the interactions of proinflammatory platelets with leukocytes during the recruitment of neutrophils and other types of leukocytes to sites of inflammation. Our lab has contributed the identification of platelet Rac-GEFs that are crucial for these interactions. I nthe absence of these platelet Rac-GEFs, platelet/leukocyte interactions are blocked and, in consequence, leukcoyte recruitment to sites of inflammation is also blocked.
Collaborator Contribution Our collaborators have contributed their esxpertise in platelet biology, pulmonary inflammation and intravital microscopy.
Impact Joint publication in Blood in 2015. Planned joint grant applications. Continued collaboration on related projects.
Start Year 2012
 
Description Simon Pitchford and Clive Page (KCL, London) 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Role of platelets in leukocyte recruitment
Collaborator Contribution joint experiments lab visits joint publications
Impact joint papers in Blood and in Curr Opin In Hematol
Start Year 2009
 
Description Takehiko Sasaki 
Organisation Akita University
Country Japan 
Sector Academic/University 
PI Contribution We have measured phopshoinositides and studied the expression of various PI3K-pathway proteins in cells or tissues prepared by the Sasaki lab. We have trained members of their lab in lipidomics methods and in measuring PIP3-dependent phosphorylation of PKB at BI and supplied them with reagents to do these assays in Akita
Collaborator Contribution Supplied cells and tissues from mouse lines or mouse models in their lab.
Impact A number of papers have been co-authored, including 1 in 2017-2018 and further papers are planned.
 
Description Vernalis Research Cambridge 
Organisation Vernalis
Country United Kingdom 
Sector Private 
PI Contribution Wrote iCase studentship application, recruited joint PhD student Elizabeth Hampson, supervision of student and carrying out of project
Collaborator Contribution Provision of time and expertise and industrial placement,
Impact Recently established collaboration, no outputs yet
Start Year 2017
 
Description Associate Editor J Immunol 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact refereeing papers
Year(s) Of Engagement Activity 2016,2017,2018,2019
 
Description Cambridge Science Festival , David Barneda 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Cambridge science festival presentation/ discussion on the role of science in society
Year(s) Of Engagement Activity 2019
 
Description Cambridge Science Festival Escape room 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We ran an escape room experience as part fo the wider Cambridge Science festival event.
Year(s) Of Engagement Activity 2018
 
Description Editor Phagocytes.EU 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Editor of networking website
Year(s) Of Engagement Activity 2019
URL https://phagocytes.eu/
 
Description Editorial board member Cell Rep 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Editorial board member of
Cell Reports
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Editorial board member Small GTPases 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Reviewing papers
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019
 
Description European Phagocyte Workshop - Member of steering committee 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Steering committee of Annual European Phagocyte Workshop
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description How to read research papers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Discussion with Bristol university PhD studetns about reading and analysing research papers.
Year(s) Of Engagement Activity 2018
 
Description Launchpad activity day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Activity day associated with launch pad initiative
Year(s) Of Engagement Activity 2018
 
Description Launchpad filming 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Part of the filming was at Babraham
Year(s) Of Engagement Activity 2018
 
Description Leader of discussion about running a successful research team with BBSRC fellows. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Discussion with BBSRC fellows about issues inovled in running research teams, strengths, weaknesses, mistakes, successes.
Year(s) Of Engagement Activity 2018
 
Description Lifelab 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The event attracted a total audience of 1765
Year(s) Of Engagement Activity 2018
 
Description Participation in Public Engagement event on genome editing - part of the Cambridge Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A debate/dialgoue open to the pulbic entitled 'Genome editing: How far should we go'
Year(s) Of Engagement Activity 2018
 
Description Phagocytes.Net 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I am Editor of this website for fellow phagocyte researchers, postdocs, students and the general public that provides information on phagocyte-related events, research groups, and jobs. www.phagcytes.net

Better communication among research groups in the phagocyte field. Markedly increased numbers of participants at the annual meetings of the European Phagocyte Workshop.
Year(s) Of Engagement Activity 2006,2008,2009,2010,2011
URL http://www.phagcytes.net
 
Description Royal society summer exhibition 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Exhibits interactively presenting the work in the signalling programme where presented at the Royal Soc, audience of approx 10000 over 7 days
Year(s) Of Engagement Activity 2018
 
Description Schools Days at Babraham 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact We host 10 GCSE or A level students for one day every year to conduct experiments.

Rasied image of "science" as a profession and of "scientists" among young people in the area.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Schools day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 125 pupils attended schools day, talks given to all and lab practicals ran for two small groups.
Year(s) Of Engagement Activity 2018
 
Description Schools day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact School children from across the country attended an event at Babraham
Year(s) Of Engagement Activity 2018
 
Description Schools day Feb 2018 Babraham Institute 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Discussion, question/answer and experiment to show how neutrophils contribute to immune defence and a broader dialogue about the societal signifcance of research and the types of jobs that can be involved.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
 
Description Work Experience students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact School students came into our department to shadow researchers and get involved in discussions about research and see our facilities.
Year(s) Of Engagement Activity 2018
 
Description schools day - Babraham 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact We provided laboratory experiments based on our research for secondary school children (GCSE and A-level). Two groups of 5-7 children attended for one day each year. Three members of my team spent a total of about 10 hrs preparing for this event and were each fully occupied on the day.

Students report that they gained a real sense of what working in scientific research is like. Schools have always reported a favourable impact on pupls decisions to pursue science at both 'A-level' and degree level and return each year.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description stem insights teachers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact STEM teachers attended an event at Babraham designed to give them insight into the process of research and careers in research.
Year(s) Of Engagement Activity 2018