The role of PtdIns3P in the killing of bacteria and fungi by the neutrophil NADPH oxidase

Lead Research Organisation: Babraham Institute
Department Name: Dept of Development & Signalling

Abstract

Neutrophils are white blood cells in our body which are responsible for finding, eating and killing nasty microbes. An important part of the killing process is the delivery of large quantities of toxic oxygen molecules into a specialist chamber inside the neutrophil called a phagosome, which contains the microbe. These toxic chemicals are made by an enzyme called the NADPH oxidase and we know it must be important because of the discovery of a rare disease called CGD (chronic granulomatous disease), where patients carrying mutations in one of the six proteins which make up this enzyme suffer life-threatening bacterial and fungal infections. The way in which the NADPH oxidase is turned on is complicated, because it has to switch on in response to eating a wide variety of different microbes but at the same time it mustn?t cause too much damage to surrounding body tissues. An understanding of how the NADPH oxidase works is thus likely to improve our understanding of what goes wrong when some infections and inflammations are not dealt with properly. The applicants have recently discovered that enzymes called PI3Ks may play a key role in regulating the NADPH oxidase by synthesising a messenger molecule around the phagosome called PtdIns3P, which they have shown binds tightly to one of the six NADPH oxidase components. They propose to use chemicals recently developed by the pharmaceutical industry to specifically block PI3Ks to assess their role in the synthesis of PtdIns3P and NADPH oxidase function. Most of these experiments will be done on neutrophils purified from human blood taken from local volunteers. The applicants have also created mutant mice in which the gene encoding the expression of the oxidase component which binds PtdIns3P has been altered to prevent this interaction. These mice are healthy and fertile when housed under pathogen-free conditions and they now propose to evaluate the effect of this mutation on the ability of these mice to combat bacterial and fungal infections; most of the experiments will be done on neutrophils purified from the bone marrow of these animals. This group has a strong track record in publishing their work in the public domain and are regularly asked to present their work at international conferences. They have also developed good links with the pharmaceutical industry and hence useful results from the work should be quickly disseminated.

Technical Summary

The NADPH oxidase of neutrophils plays a key role in our defence against microbial pathogens. This is clearly evident from the life-threatening bacterial and fungal infections which present in cases of CGD, a disease caused by mutations in several of the components of this enzyme complex. The NADPH oxidase is responsible for directing the delivery of toxic reactive oxygen species into phagosomes where they play an important role in the killing of engulfed pathogens. The signalling pathways which lead to the appropriate assembly and activation of the oxidase are necessarily complex, both because of the wide variety of receptors which are required to trigger this event and the need for tight control to limit damage to host tissues. This proposal seeks to understand the role of phosphoinositide 3-kinase (PI3K) signalling pathways in NADPH oxidase activation and killing of specific bacteria and fungi.

There are seven different catalytic subunits of PI3Ks which fall into three functional classes; of relevance here are the class I and class III enzymes which synthesise the phospholipid messengers PtdIns(3,4,5)P3 and PtdIns3P, respectively. A major objective of this proposal is to use newly developed PI3K isoform-selective inhibitors and PI3K ?knock-out? mice to define the involvement of specific PI3Ks in human and mouse neutrophil phagocytosis, NADPH oxidase activation and killing of specific bacteria and fungi. The majority of these analyses will be carried out in vitro on neutrophils purified from human blood and mouse bone marrow. A second objective will be to measure PtdIns3P synthesis and subcellular localisation during selected examples of phagocytosis by confocal fluorescence imaging of neutrophils expressing GFP-tagged, PtdIns3P-binding domains. A third objective will be to measure the effects of a mutation in one of the components of the oxidase, p40phox, which prevents binding to PtdIns3P; this will be done by utilising p40phox R58A ?knock-in? mice recently created in the applicants? laboratory and will involve characterising NADPH oxidase responses in vitro and killing of bacteria and fungi both in vitro and in vivo.

It is anticipated that the proposed work will define some important paradigms for how PI3Ks are used to regulate NADPH oxidase activity during infection and inflammation and the applicants are well placed to disseminate this information through publication in peer reviewed journals, presentations at scientific conferences and through good collaborations with the pharmaceutical industry.
 
Description post-doctoral training award/Roberts Funding
Amount £1,000 (GBP)
Organisation Babraham Institute 
Sector Academic/University
Country United Kingdom
Start 09/2010 
End 10/2010
 
Title Generation of p40phoxR58A knock-in mice 
Description The generation by gene-targetting of a strain of mice in which both alleles of the p40phox subunit of the NADPH oxidase harbour an R58A knock-in mutation. This mutation blocks the interaction of this subunit with PtdIns3P, the lipid product of type III PI3K. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2008 
Provided To Others? Yes  
Impact PMID: 20813901 and PMID: 18755982 listed under 'publications' 
URL http://europepmc.org/abstract/MED/20813901
 
Title Neutrophil activation by Aspergillus Fumigatus hyphae 
Description Assays were developed to measure neutrophil activation upon recognition of the hyphal form of the fungus Aspergillus Fumigatus. These included assays for the production of reactive oxygen species and the activation of PI3K signalling pathways. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Boyle et al (2011) J Immunol. 186(5):2978-89. doi: 10.4049/jimmunol.1002268. PMID: 21257963 
 
Description Mocsai - neutrophil biology 
Organisation Semmelweiss University
Country Hungary 
Sector Academic/University 
PI Contribution expertise and reagents
Collaborator Contribution expertise, reagents and exchange of personnel
Impact Gyori et al (2014) Arthritis Rheumatol. 66(8):2210-21. doi: 10.1002/art.38660 Boyle et al (2011) J Immunol. 186(5):2978-89. doi: 10.4049/jimmunol.1002268. Research Grant from Karus Therapeutics, UK
Start Year 2010
 
Description Role of CD18 in neutrophil oxidase activation by bacteria and fungi 
Organisation University of Ulm
Department Department of Dermatology and Allergic Diseases
Country Germany 
Sector Academic/University 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution Access to CD18 knock-out mice
Impact PMID: 18755982 listed under 'publications'.
Start Year 2007
 
Description Role of Dectin-1 in neutrophil activation by Aspergillus 
Organisation Cardiff University
Department School of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We took the intellectual lead in this project and contributed the majority of the work and reagents, including strains of mice deficient in PI3K and NADPH oxidase subunits
Collaborator Contribution access to Dectin-1 knock-out mice plus intellectual contribution to project
Impact Boyle et al (2011) J Immunol. 186(5):2978-89. doi: 10.4049/jimmunol.1002268.
Start Year 2009
 
Description Role of Rab27 in neutrophil oxidase activation 
Organisation Imperial College London
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution Provided access to Rab27 knock-out mice
Impact PMID: 20813901 listed under 'publications'
Start Year 2009
 
Description Role of Rac proteins in neutrophil oxidase activation by bacteria 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution Provided access to Rac1 knock-down and Rac2 knock-out mouse strains
Impact PMID: 20813901 listed under 'publications'
Start Year 2007
 
Description Role of Rac proteins in neutrophil oxidase activation by bacteria 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution Provided access to Rac1 knock-down and Rac2 knock-out mouse strains
Impact PMID: 20813901 listed under 'publications'
Start Year 2007
 
Description Role of Syk in neutrophil activation by Aspergillus 
Organisation Semmelweiss University
Department Department of Physiology
Country Hungary 
Sector Academic/University 
PI Contribution We took the intellectual lead in this project and contributed the majority of the work and reagents, including strains of mice deficient in PI3K and NADPH oxidase subunits
Collaborator Contribution Our collaborators provided access to two strains of knock-out mice (syk KO; Fcgamma/DAP12 double KO) and hosted a visit from Keith Boyle (post-doctoral scientist employed on grant) to their laboratory in Budapest to conduct experiments.
Impact Boyle KB et al (2011) J Immunol. 186(5):2978-89. doi: 10.4049/jimmunol.1002268.
Start Year 2009
 
Description Role of Syk in neutrophil activation by Aspergillus 
Organisation Semmelweiss University
Country Hungary 
Sector Academic/University 
PI Contribution We took the intellectual lead in this project and contributed the majority of the work and reagents, including strains of mice deficient in PI3K and NADPH oxidase subunits
Collaborator Contribution Our collaborators provided access to two strains of knock-out mice (syk KO; Fcgamma/DAP12 double KO) and hosted a visit from Keith Boyle (post-doctoral scientist employed on grant) to their laboratory in Budapest to conduct experiments.
Impact Boyle KB et al (2011) J Immunol. 186(5):2978-89. doi: 10.4049/jimmunol.1002268.
Start Year 2009
 
Description Role of the Fc gamma chain in neutrophil oxidase activation by bacteria 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution provided access to Fc gamma chain knock-out mice
Impact PMID: 18755982 listed under 'publications'
Start Year 2007
 
Description UCB pharma studentship 
Organisation UCB Pharma
Country United Kingdom 
Sector Private 
PI Contribution We provided the majority of the intellectual input and technical resources for this collaboration
Collaborator Contribution funded a PhD studentship that contributed to the project, also provided reagents and intellectual input
Impact PMID: 20813901 and PMID: 18755982 listed under 'publications'
 
Description Babraham Village Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact About 30 local parishioners attended a Q&A session at the Babraham Institute

I believe this activity helped inform local inhabitants about the medical research conducted at the Babraham Institute and hence helped to develop their opinions concerning local planning applications and also the activities of animal rights protestors.
Year(s) Of Engagement Activity 2007
 
Description Invited lecturer to international meetings (average 2-3 per year) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact promoted discussions, collaborations

scientific collaborations, joint grants and publications
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description babraham lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Explaining our work to non-academic employees at the Babraham Institute through a formal lecture

a wider appreciation of MRC funded science at the Babraham Institute
Year(s) Of Engagement Activity 2008
 
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 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019