The Mechanisms of TLR-Mediated Regulation of Neutrophil Survival

Lead Research Organisation: University of Sheffield
Department Name: Medicine and Biomedical Science

Abstract

There is a large group of lung diseases that are both common and hard to treat. Their unifying feature is that they are all caused by the over-activation of a white blood cell called the neutrophil. In general, neutrophils are hugely important. They are very numerous in the blood, and are mobilised very quickly to sites of infection, where they hunt and destroy bacteria, fungi, and some viruses. Their ability to kill bugs comes from carrying a range of toxic proteins which they deliberately release onto the microbes. When the neutrophil?s job is done, it naturally dies and is removed by cells specialised in clearing up (like macrophages). The death of the neutrophil is highly regulated in a process called apoptosis, and is designed so that the neutrophil doesn?t spill any of its toxic antimicrobial compounds into healthy or healing tissues. However, if the neutrophil lives too long, or doesn?t get cleared properly when it dies, spillage of these toxic compounds can cause major tissue damage. This is thought to be an important contributor to a wide range of diseases, for example chronic obstructive pulmonary disease (COPD, commonly caused by smoking).
We think we?ve worked out how neutrophil survival might be regulated at sites of infection. This survival response depends upon a clever series of orchestrated signals that keep the neutrophil alive for just the right length of time. We also think that, in diseases like COPD, these survival signals are doing too much. Our work plans on studying these systems in much more detail. We want to look at normal neutrophils, and neutrophils taken from the lungs during inflammation. To study these pathways in the test tube, we plan to make neutrophils from stem cells that we have manipulated to be deficient in specific proteins. Putting together these approaches, many of which are really new and technically very exciting, we think we?ll identify some genuinely new potential drug targets that might revolutionise the treatment of lung disease.

Technical Summary

The neutrophil is our most numerous phagocyte and a principal line of antimicrobial defence, but where activated inappropriately, neutrophilic inflammation is a major cause of airways disease and is very difficult to treat.
Neutrophils are short-lived cells, whose death by apoptosis allows resolution of inflammation once infection has been removed. We have shown that neutrophil lifespan is extended through activation of a major group of pathogen and damage recognition receptors, the TLRs. At sites of inflammation, infectious stimuli and signals released from damaged tissues activate TLRs to cause a delay in apoptosis that facilitates clearance of infections.
We have now shown that TLR-mediated neutrophil survival is a two-stage process, regulated by independent signalling pathways. First, direct activation of TLRs on the neutrophil results in transient induction of neutrophil survival. Secondly, activation of TLRs on other cells present at sites of inflammation, such as monocytes, releases survival signals that cause prolonged neutrophil survival. These indirect survival signals act on neutrophil cAMP signalling, and may explain why drugs acting to raise cAMP can fail to control neutrophilic inflammation.
We propose that the handover of regulation of neutrophil survival to external control allows for the withdrawal of survival stimuli and facilitates resolution of injury when danger is passed.
We hypothesise that the targeting of these pathways would restore normal rates of neutrophil apoptosis in inflammatory diseases.
We propose to study, in vitro and in vivo, both components of the regulation of neutrophil survival. First, we will determine how the direct infection signal (mediated by activation of TLR4) is limited in its efficacy, since this may be dysregulated in disease, and represent a new therapeutic target. Secondly, we will use a combination of microarray and proteomic approaches to determine how cAMP-mediated signalling causes neutrophil survival. We will challenge human subjects with inhaled endotoxin, and explore in vivo how these direct and indirect survival signals contribute to the regulation of neutrophilic airway inflammation. Neutrophils are hard to genetically modify, so when we identify new targets involved in the regulation of these survival pathways, we will knock down their function in neutrophils derived from bone marrow and embryonic stem cells, using methodology established in our groups. These studies will identify new pathways, and hence therapeutic targets, regulating neutrophil apoptosis in man, and target their function using novel approaches to the study of neutrophil biology.
 
Description Heart Research UK Fellowship
Amount £150,000 (GBP)
Organisation Heart Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2012 
End 02/2014
 
Description MRC ABPI COPD initiative
Amount £150,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2011 
End 12/2013
 
Description MRC PhD studentship
Amount £35,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 02/2015
 
Description NIHR Fellowship
Amount £30,000 (GBP)
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 04/2012 
End 03/2014
 
Description COPD MAP 
Organisation Medical Research Council (MRC)
Country United Kingdom 
Sector Public 
PI Contribution Expertise in neutrophil biology and pathogenesis of inflammation
Collaborator Contribution Access to additional patient samples and tool compounds from Industry
Impact Funding but no publication outputs as yet; new collaboration
Start Year 2011
 
Description Collaboration for bioinformatic approaches 
Organisation University of Liverpool
Department School of Biological Sciences Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed detailed collaboration to facilitate bioinformatic approach to neutrophil apoptosis
Collaborator Contribution Provided analysis techniques resulting in new hypothesis development
Impact Publications currently in preparation
Start Year 2011
 
Description GSK collaboration for access to compounds 
Organisation GlaxoSmithKline (GSK)
Department Respiratory Development Planning Unit DPU
Country United Kingdom 
Sector Private 
PI Contribution Discussed data regarding mechanisms of neutrophil apoptosis
Collaborator Contribution Provision of reagents and intellectual contribution
Impact Publication in preparation
Start Year 2011
 
Description University of Graz 
Organisation University of Graz
Department Pharmacology
Country Austria 
Sector Academic/University 
PI Contribution Collaborative exploration of prostaglandin receptor expression and function in neutrophils
Collaborator Contribution Advice regarding antibodies and compounds, provision of collaborative data
Impact None as yet, expecting to contribute to next manuscript
Start Year 2013
 
Description Zebrafish 
Organisation University of Sheffield
Department Department of Infection, Immunity and Cardiovascular Disease
Country United Kingdom 
Sector Academic/University 
PI Contribution Shared knowledge re PKA biology
Collaborator Contribution Set up zebrafish models of key neutrophil regulatory pathways identified by our research
Impact Data to contribute to next manuscript
Start Year 2012
 
Description Asthma and airway inflammation national science week presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Spoke to a whole year group (approx 120 children) of late teenage on the biology of asthma, took members of research group to explain science, generated interest in pathobiology of asthma

None specific
Year(s) Of Engagement Activity 2010
 
Description National Science Week School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Approx 120 students aged 14-16 attended a talk on airways inflammation and asthma, given by me and my group (including LCP who was funded by this fellowship).

Generated interest by school children in asthma and inflammation
Year(s) Of Engagement Activity 2010
 
Description Primitive Streak 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact The Primitive Streak Exhibition (Helen Storey Foundation) was used as a vehicle for a national science week public lecture with demonstrations related to the lung. There were also opportunities in the central Winter Gardens in Sheffield for the public to have their spirometry measured and understand more about how the lungs work. A series of special dresses were created by Helen Storey to illustrate the function of the lung, as part of her longstanding interest in using new techniques to explain human biology.

Local and national press activity
Year(s) Of Engagement Activity 2011
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Talked to 60 primary school children about lung science and health

Asked for repeat visit each year
Year(s) Of Engagement Activity 2012
 
Description School visit through National Science Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact We attended a local primary school, and engaged in workshops with the whole year group (about 80 pupils) to explain how airway inflammation works.

We have been asked to do this again, received excellent feedback
Year(s) Of Engagement Activity 2011
 
Description Talks at International Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact 'Targeting TLRs'. New Drugs for Asthma and COPD, Brompton Hospital 2008. 'The networks that regulate innate and adaptive immunity in asthma and COPD'. Invited talk for the joint BTS/BALR symposium at the British Thoracic Society Winter meeting, London 2007. 8th Annual Lund COPD Symposium April 2007 talk title 'Integration of the innate and adaptive immune responses in COPD'. Chair of Session and speaker at British Society for Allergy and Clinical Immunology, July 2007. Talk title 'Inflammatory Networks of Asthma' 'Toll-like receptors:  Potential therapeutic aspects'. Joint British Thoracic Society/ British Society for Allergy and Clinical Immunology, BTS Winter meeting, Dec 2006

Generated useful collaborations and raised importance of field
Year(s) Of Engagement Activity 2006,2007,2008