MICA: Finding new treatments for failed resolution of inflammation using zebrafish models.

Lead Research Organisation: University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease

Abstract

Neutrophils are a type of white blood cell that are important in fighting infection and healing wounds (in a process called inflammation). We know that neutrophils need to be removed when their job is done or they can cause damage. The mechanisms of neutrophil removal are particularly important in a delicate organ such as the lung where failure can mean permanent lung damage as is seen in Chronic Obstructive Pulmonary Disease (COPD), a common disabling disease with no cure. We do not understand how neutrophils know when the job is done and how this translates into signals to remove neutrophils from their place of work - the inflammatory site. In my lab, I have developed tools in zebrafish that allow me to see neutrophils as they fight infection, heal wounds and importantly as they are removed during healing (or "resolution") of inflammation. These zebrafish can be genetically modified to express glowing (fluorescent) proteins that label neutrophils or to change the genes controlling inflammation. This allows us to get a much better understanding of the processes and controls of inflammation resolution. With these models, we have seen how neutrophils can be removed in 2 ways, by moving away from the site of inflammation or by dying there and being removed by another type of immune cell (macrophages).
In this programme, I propose to use zebrafish models and human neutrophils to understand the signals keeping neutrophils at sites of inflammation, which I call "retention signals". I have identified a drug that works by causing neutrophils to leave sites of inflammation and I will investigate how this changes the retention signals that neutrophils use to keep them at inflammatory sites. I will then study the downstream signals within neutrophils that act to turn these signals into changes in behaviour of the neutrophils. From recent studies in my lab, I have identified two important pathways that might be key regulators of retention signalling. The first is an enzyme, Serum and Glucocorticoid regulated kinase-1 (SGK1) that seems to act to retain living neutrophils at sites of inflammation. I will explore the potential of this kinase and related molecules to control inflammation resolution. Since this is a good drug target, this aim will be explored in collaboration with GlaxoSmithKline who are working with me to identify drug candidates targeting this molecule. Finally, a gene called Kalirin was identified in a genetic screen in my lab as a regulator of inflammation resolution. Kalirin is a protein normally controlling the direction of nerve cell growth, but it is present in neutrophils where it might control neutrophil movement. I will explore the possibility that Kalirin is involved in retention signalling and see whether it might be a suitable target for drug discovery.
Taken together these approaches will uncover new aspects of the control of inflammation resolution and identify a number of targets for drug discovery. I hope this will ultimately find new ways to treat inflammatory diseases.

Technical Summary

Diseases of failed inflammation resolution such as COPD are major causes of morbidity and mortality. Currently no therapies target the key effector cell, the neutrophil, to drive inflammation resolution. Targeting neutrophil recruitment leaves tissues unprotected against infection, and new approaches are needed to remove unwanted neutrophils without impacting host defence. We are in the midst of a revolution in neutrophil biology, in which neutrophils are recognised to be more long-lived, with active roles in programming adaptive immunity and regulating immune homeostasis. Neutrophils are now recognised to have a number of potential fates in addition to apoptosis. Neutrophils can reverse transmigrate back into the circulation in humans and mice, they can be lost into exudates in the mammalian lung or they can reverse migrate within tissues to dissipate the inflammatory burden - shown in zebrafish and with human neutrophils.
I hypothesise that neutrophils are retained at inflammatory sites by "retention signals" that delay reverse migration, and that neutrophil sensitivity to these signals is mediated through a pathway requiring Serum and Glucocorticoid-regulated Kinase-1 (SGK1) and Kalirin, providing a series of new targets for drug discovery. In this Programme, I will identify the nature of retention signals and how these overlap with survival signals, then explore the molecular mechanism of action of Tanshinone IIA (identified by drug screening in my lab) which drives reverse migration. I will define the functional neutrophil kinome and exploit novel kinase inhibitors and kinase targets for the selective removal of inflammatory neutrophils, using SGK1 as a prototypic downstream signal important in retention signalling. Finally, I will define the role of Kalirin (identified by genetic screening in my lab), as a regulator of neutrophil retention signalling.

Planned Impact

The mechanisms regulating inflammation resolution are of fundamental importance in immunology. Very few targets with critical roles in inflammation resolution have been identified to date. The discoveries from this project will therefore significantly enhance the knowledge economy with new scientific advancement, as described in 'academic beneficiaries".
There is considerable interest from the pharmaceutical industry in discovering regulators of inflammation resolution, which are fundamental in a range of disease settings. In my own clinical speciality of respiratory medicine, Chronic Obstructive Pulmonary Disease is a major cause of morbidity and mortality which lacks effective treatments. Many examples exist in all clinical specialities. We anticipate that the unique nature of the mechanisms investigated in this Programme will make them attractive targets for anti-inflammatory therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as the MICA collaboration with GlaxoSmithKline for Aim 2 of this proposal.
It is my ultimate aim that drugs identified in my research programme find their way into my clinical practice to treat patients who I currently cannot offer effective treatments. This programme will deliver successful identification of drug targets and identification of lead candidates which I hope will ultimately have an impact on patient care. The approaches I have developed are suitable for drug repurposing, and identification of compounds such as Tanshinone IIA (used in China to treat cardiovascular disease) shows how this might be possible. Experimental Medicine approaches are in development to take forward such advances towards clinical use. The project will validate approaches for the identification of new small molecular inhibitors of retention signalling in inflammation. This leads to obvious longer-term commercial opportunities to develop the technology to a higher throughput level, and to engage industrial partners in developing new therapies. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how.
The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being. Avoidance of dysregulated inflammation is a prerequisite for healthy ageing.
The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and molecular biology. These methodologies will be used to develop and make use of innovative approaches to the study of inflammation in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
This proposal will deliver highly trained researchers offering unique skills. The PDRAs will combine skills in fish models and state of the art in vivo microscopy, in parallel with human primary cell-based assays. They will develop distinctive skills in generating new datasets and new approaches to understanding biological problems. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies.
The PDRAs and myself are actively involved in public engagement and broader dissemination, with regular school visits and high-level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.

Publications

10 25 50

 
Description BIA Fellowship to Robbie Evans
Amount £66,460 (GBP)
Organisation British Infection Association (BIA) 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2015 
End 11/2016
 
Description Developing an in vivo CRISPR-interference Screening Resource
Amount £428,223 (GBP)
Funding ID BB/R015457/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 08/2020
 
Description Discovery Medicine North DTP in Discovery Medicine across the Lifecourse
Amount £3,300,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 01/2021
 
Description ELA International Fellowship (NH)
Amount £240,000 (GBP)
Organisation European Leukodystrophy Association (ELA) 
Sector Charity/Non Profit
Country European Union (EU)
Start 12/2016 
End 12/2019
 
Description IMI
Amount € 24,000,000 (EUR)
Organisation European Commission 
Department Innovative Medicines Initiative (IMI)
Sector Multiple
Country European Union (EU)
Start 08/2017 
End 08/2022
 
Description MRC Antimicrobial Resistance Networks
Amount £2,800,000 (GBP)
Funding ID MR/N02995X/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 08/2021
 
Description MRC CIMA studentships
Amount £120,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 10/2022
 
Description MRC Project Grant
Amount £412,769 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 12/2022
 
Description MRC Project Grant
Amount £691,917 (GBP)
Funding ID MR/R001111/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2018 
End 01/2021
 
Description NC3Rs Pilot Grant
Amount £98,500 (GBP)
Funding ID NC/M001490/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Private
Country United Kingdom
Start 03/2015 
End 10/2016
 
Description Wellcome Trust Clinical Research Training Fellowship
Amount £249,928 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Research Training Fellowship
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description Wellcome Trust Clinical Training Fellowship
Amount £250,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2021
 
Title Lyz:Halo 
Description Lyz:Halo tag transgenic zebrafish line 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact In progress 
 
Title lyz:dCas9 
Description lyz:dCas9 transgenic zebrafish line 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact In progress 
 
Title mpeg:dCas9 
Description mpeg:dCAs9 transgenic zebrafish line 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact In progress 
 
Description Future Transplantation Techniques - Zon 
Organisation Harvard University
Department Department of Stem Cell and Regenerative Biology
Country United States 
Sector Academic/University 
PI Contribution We provided images showing human cells could transplant the same way as zebrafish cells in the developing zebrafish host, thus confirming previous data obtained by Zon's team.
Collaborator Contribution Provided us with technical help when developing the technique.
Impact None yet.
Start Year 2016
 
Description Il-34 in zebrafish 
Organisation University of Sheffield
Department Department of Oncology and Metabolism
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in zebrafish mutagenesis and imaging, inflammation
Collaborator Contribution Expertise in IL-34 biology
Impact PhD student working on generating and characterising fish mutants
Start Year 2015
 
Description Infection in cystic fibrosis models Laurent Kremer 
Organisation French Institute of Health and Medical Research
Country France 
Sector Public 
PI Contribution Provided expertise in zebrafish innate immunity and modelling of infection in zebrafish, clinical insights into CF.
Collaborator Contribution Expertise in mycobacterial infection.
Impact https://doi.org/10.1016/j.celrep.2019.01.071 Marie Sklowdowska-Curie Intraeuropean Fellowship to Audrey Bernut.
Start Year 2016
 
Description Inflammation in ATM mutants 
Organisation University of Sheffield
Department Department of Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Inflammation expertise
Collaborator Contribution Neuroscience expertise
Impact In progress
Start Year 2017
 
Description Jim Wild 
Organisation University of Sheffield
Department Faculty of Medicine, Dentistry and Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided clinical input to collaborative project and clinical academic guidance to supervised clinician scientist.
Collaborator Contribution Provided MRI physics and imaging expertise to partnership.
Impact PMID: 30389827 Grant funding from IMI programme of Horizon 2020
Start Year 2015
 
Description Larger blood volume for transplant studies - Snowden 
Organisation University of Sheffield
Department School of Clinical Dentistry Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution NA
Collaborator Contribution Offered to include our study in his ethics to allow access to larger volume of human blood to perform larger experiments
Impact None yet.
Start Year 2016
 
Description MRI imaging as a biomarker for Interstitial Lung Disease 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in ILD, academic clinical mentoring of Fellow
Collaborator Contribution Expertise in MRI imaging
Impact IMI TRISTAN.Multi-disciplinary. Physics, medicine
Start Year 2015
 
Description Microglia and GBA deficiency 
Organisation University of Sheffield
Department Department of Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of expertise and transgenic lines for the study of microglia.
Collaborator Contribution Mutant zebrafish and characterisation of mutants.
Impact Manuscript in Human Molecular Genetics.
Start Year 2015
 
Description Neutrophil Kinome - Lynne Prince 
Organisation University of Sheffield
Department Faculty of Medicine, Dentistry and Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Biology of zebrafish neutrophils, screen of compounds against zebrafish neutrophilic inflammation. Characterisation of the neutrophil kinome.
Collaborator Contribution Biology of human neutrophils, screen of compounds against human neutrophil apoptosis.
Impact Manuscript in revision.
Start Year 2016
 
Description Neutrophil viscosity in vivo 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed transgenic reagent for measuring neutrophil viscosity in vivo
Collaborator Contribution Provided essential scientific input and guidance
Impact In progress
Start Year 2016
 
Description PI3K in fish 
Organisation University of Sheffield
Department Department of Infection, Immunity and Cardiovascular Disease
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in Zebrafish as a model of immunity
Collaborator Contribution PI3K biology
Impact Wellcome Clinical Research Training Fellowship. BHF grant to my collaborator.
Start Year 2015
 
Description Staphylococcus aureus host-specificity - JAG van Strijp 
Organisation University Medical Center Utrecht (UMC)
Department De Hoogstraat Rehabilitation Centre
Country Netherlands 
Sector Hospitals 
PI Contribution Provided zebrafish expertise to collaborative endeavour to generate transgenic zebrafish expressing humanised targets for staph aureus toxins.
Collaborator Contribution Provided expertise on staphyloccal toxins.
Impact PMID: 30625113 PMID: 30013237 Other manuscripts in revision.
Start Year 2014
 
Description Stefan Marciniak 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Idea that zebrafish might be an ideal system to test whether neutrophil viscosity might alter in vivo.
Collaborator Contribution Bodipy rotor, HaloTag combination to read out viscosity in vivo.
Impact Awaited.
Start Year 2016
 
Description The neutrophil kinome 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Provision of in vivo models and assays. Hypothesis to be tested.
Collaborator Contribution Kinase pharmacology expertise, compounds, analysis.
Impact MICA component to Programme grant funding.
Start Year 2014
 
Description Zebrafish models of Senescence 
Organisation University of Sheffield
Department Department of Oncology and Metabolism
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in zebrafish transgensis, inflammation and imaging.
Collaborator Contribution Expertise in senescence
Impact PhD student, funded by BBSRC
Start Year 2015
 
Description Discovery Night (Sheffield) 
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 Earn your zebrafish stripes - Members of the public were invited to earn their stripes by creating visual delights and going on a zebrafish tour of the world. The Bateson Centre Zebrafish Facility has the capacity to hold over 112,000 fish and is one of the largest facilities in the UK. Visitors found out why we keep so many tropical freshwater fish and explored the relevance of zebrafish in medical research at an interactive exhibition.
Year(s) Of Engagement Activity 2016
URL http://www.sheffield.ac.uk/discoverynight/index
 
Description Invited speaker - Max Planck Institute for Infection Biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Invited speaker to the Max Planck Institute for Infection Biology 'Neutrophil migration patterns in regulating inflammation'. Berlin, Germany, January 2019.
Year(s) Of Engagement Activity 2019
 
Description Krebs Festival 
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 Demonstrated how scientists at the Bateson Centre work with zebrafish to study a range of diseases.
Used microscopes to observe the transparent embryos, and see the heart pumping and blood circulating.
Year(s) Of Engagement Activity 2015
URL http://krebsfest.group.shef.ac.uk/programme/public-night/
 
Description Life: A Festival of Health, from Head to Toe, Our Microscopic Army Against Disease (Sheffield) 
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 Members of the public were shown how we use flies, fish and amoebae to understand the biology of white blood cells during disease. Activities included: fluorescence microscopy of the live organisms, 3D-fluorescent printed models of each organism, informative posters, timelapse microscopy videos and handouts.
Year(s) Of Engagement Activity 2016
URL http://microarmy.weebly.com/
 
Description Life: A Festival of Medicine, Dentistry and Health 
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 presented an exhibition, demonstrating how zebrafish can help us understand human disease. Members of the public took part in a range of activities, including looking at live transgenic zebrafish through the microscope.
Year(s) Of Engagement Activity 2014
 
Description Symposium presentation at American Thoracic Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Symposium presentation at American Thoracic Society, Denver, May 20th 2015. Invited Speaker. New insights into neutrophilic pulmonary inflammation.
Year(s) Of Engagement Activity 2015
 
Description ZDM8 Boston MA. September 2015 Invited Speaker. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact ZDM8 Boston MA. September 2015 Invited Speaker.
Year(s) Of Engagement Activity 2015