Elucidating the local site and cell types involved in antigen uptake, processing and presentation in the chicken

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

The blood and tissues of an adult organism consist of a number of cell types, including the red blood cells that carry oxygen and immune cells that fight disease. Immune cells respond to infection by viruses or microbes and initiate cellular and molecular defence mechanisms, known as an "immune response". An understanding of how the immune system defends the body against diseases is critically important if we want to develop disease control measures, such as vaccination. Most research on the immune response has been carried out on humans and in animal models such as mice. However, it is very important that we understand how the immune system of farm animals works. A key example of this is the chicken: the chicken is the most popular food animal on the planet, with over 50 billion being hatched and raised each year. Control of diseases is a major challenge in chicken production in terms of economic cost and animal welfare. An additional factor is that some bird diseases, such as bird flu, can potentially infect humans and are therefore a major challenge to human health. Protecting the poultry industry in terms of food security, public health and consumer safety is of paramount importance, not only in the UK, but worldwide. One complicating factor in increasing our understanding of the chicken's immune system and immune response to disease is that the bird's immune system is quite different to that of mammals. Some of the organs specialised for immune function in mammals, such as lymph nodes, are not present in birds, whereas birds have their own specialised immune organs that are not present in mammals. Some immune cells are similar between mammals and birds, but it is not known if these share the same functions or have different requirements for generation of an effective immune response. One of the most important types of immune cells are mononuclear phagocytes. These are a class of cells with many different functions and consequently there are many different subsets of these cells. One very important function of mononuclear phagocytes is to process antigen material and present it to other cells of the immune system, which can then respond appropriately. In this sense they communicate information on the nature of a potential pathogen to the rest of the immune system. There is very little information about what cell type(s) capture, process and present antigen in birds. We need to gain more information on these processes and we have developed a new tool for studying these mononuclear phagocytes by making transgenic chickens (called the "MacReporter" chicken) that express fluorescent proteins in mononuclear phagocytes. During our initial characterisation of the transgenic birds we found that not only could all mononuclear phagocytes be identified, but we could also identify entire immune-specific tissues. This is particularly interesting as this means that we can use these transgenic chickens to describe the structure of these immune tissues as well as analyse mononuclear phagocytes specifically. The main objectives of this research proposal are to use the MacReporter chicken to (1) identify cells and tissues involved in antigen uptake and processing in the chicken spleen, respiratory and gastrointestinal tracts; (2) identify and functionally characterise chicken splenic antigen presenting cells; (3) determine which genes are specifically expressed in the different classes of mononuclear phagocytes to further define and understand their function. In the longer term this knowledge will inform the development of better vaccines to prevent disease in chickens with the aim of improving production costs, biosecurity and welfare of production chickens.

Technical Summary

Development of more efficient vaccines will require concomitant development of reagents and functional assays to assess immune function and thereby improved understanding of both the sites and mechanisms of antigen presentation in the chicken. Full characterisation of both the major immune cell types associated with antigen uptake, processing and presentation, and the lymphoid structures in which these processes take place, will underpin this.
Objective 1: To identify cells and tissues involved in antigen uptake and processing in the chicken gastrointestinal and respiratory tracts, and the spleen. We will use our unique MacReporter chicken line, in which expression of a fluorescent reporter protein is restricted to cells of the mononuclear phagocyte lineage. Tagged antigens will be administered by the intra-tracheal, intra-cloacal and intra-venous routes to determine the structures in which antigen is taken up and processed, and which cells act as antigen-presenting cells (APC). The phenotypes of cell subsets associated with antigen sampling, processing, and identification of the lymphoid tissue compartments in which these processes take place will be established. Candidate chicken APC populations will be identified, whose ability to stimulate naïve T cells will be tested in objective 2.
Objective 2: To functionally characterise chicken APC. The aim is to test the ability of purified candidate chicken APC (obj. 1) to present antigen to naïve T cells in vitro. Potential polarisation of T cells to specific functional subsets (Th1, Th2, Th17 and Treg) will be determined by qPCR and ELISA.
Objective 3: To characterise the gene expression profiles of cell subsets associated with antigen uptake, processing and presentation. Once the APC are characterised (obj. 1 and 2), cell subsets will be purified by FACS and their gene expression profiles characterised by RNASeq to identify the gene expression signatures associated with antigen sampling, processing and presentation.

Planned Impact

The work proposed has direct relevance to the strategic priorities of the BBSRC - Animal Health. Infectious diseases are a constant threat to the poultry industry through losses or reduction in production, decreases in egg production and quality, and effects on animal welfare. Vaccines are used to control the major diseases of poultry but the immunological mechanisms that lead to success or failure of novel and current vaccines have not been elucidated and improvement has been hampered by the lack of fundamental knowledge of the chicken's mucosal immune system. The ability to combat infectious diseases which reduce the health and welfare of farm animals requires more efficient vaccines, reagents and functional assays to assess immune function and the need to understand both the sites and mechanisms of antigen presentation in the chicken.
Outputs of the work proposed will include the identification of cell subsets associated with antigen uptake, processing and presentation. These antigen presenting cells are major players in the induction of immune responses and are the target cells to improve vaccine responses. Outcomes will provide crucial information for the development of more efficient vaccines, ensuring that poultry farming remains not only a secure food source but also increases the economic competitiveness of the UK.

The following stakeholders have been identified as beneficiaries of this work:

The poultry production industry
Infectious diseases cost the UK poultry industry billions of pounds per annum. Understanding vaccine responsiveness and resistance to diseases in poultry improves the efficiency of the industry, through improved protection against diseases and the development of more efficient vaccines.

The poultry breeding industry
The consequences of improved vaccine responses and disease resistance may provide a panel of phenotypic biomarkers which could be developed as affordable tools to inform breeding strategy. We have established collaborations with major poultry breeding companies.

The animal health industry
The RI has established collaborations, including direct support, with several vaccine companies that have resulted in ongoing assessment of potential vaccine candidates and immunomodulatory products. The data generated during this project will allow us to improve vaccine targeting and will develop tools to modulate immune responses at mucosal surfaces.

Animal welfare
The reduction of disease as a result of improved vaccine strategies supports the Five Freedoms implicit to animal welfare as set out by the Farm Animal Welfare Council.

General public and the environment
The consequences of improved vaccine responses and disease resistance will lead to a reduction in the prophylactic use of antimicrobials and the risk of contamination of the food chain and the environment.

Academia and Training
The multidisciplinary nature of this project will provide opportunities for broad training to all staff including other members and students of the institution ('strengthen the research community in the areas of disease and pest resistance of farmed animals through interdisciplinary research and the provision of training'). Results with respect to the identification of cell subsets associated with antigen uptake, processing and presentation will be of interest to a wide scientific community and will be published in peer-reviewed journals and presented at national and international scientific meetings.
 
Description We have discovered novel sub-populations of antigen presenting cells in the chicken spleen (a systemic organ) and in mucosal tissues including the respiratory tract. These subpopulations cells have been sorted using FACS and transcriptome analysis of the sub-populations performed.
Assays to investigate functionality of the various sub-populations of antigen presenting cells have been developed and include quantification of phagocytosis and break down of proteins and acidification assays.
Specialised cells in the epithelium of the mucosal tissues, M cells, transport molecules and microbes across the epithelium into the tissue. We have identified and characterized these cells in the chicken. Further research will investigate if these cells can be used to improve to uptake of vaccines through specifically targeting vaccines to their surface.
Exploitation Route This will be discussed with industrial partners to explore mucosal vaccine targeting.
Our most recent findings have led to further funding; BBSRC FTMA, MRC IVVN and an Innovate UK grant in collaboration with Vaxcine/Proxima Concepts Ltd.

The work has also led to the funding of a PhD studentship; Enlightenment Scholarship funded by UoE.
Sectors Agriculture, Food and Drink

 
Description The findings from this award have led to interest from pharmaceutical industry. We are discussing possibilities for follow up research in which the industry will participate. This will allow the early career scientist on the project to gain transferable skills and interact with industry.
First Year Of Impact 2017
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description BBSRC Flexible Talent Mobility Award
Amount £12,330 (GBP)
Funding ID BB/R506564/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description BBSRC Responsive Mode
Amount £471,945 (GBP)
Funding ID BB/R003653/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 11/2020
 
Description Research Grant - Lonneke Vervelde - Towards edible vaccines for chickens
Amount £99,656 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 12/2019
 
Description Roslin Foundation Avian Biology and Quantitative Genetics PhD studentships
Amount £100,000 (GBP)
Organisation The Roslin Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Description Sub-award from BBSRC Flexible Talent Mobility Award (BB/R506564/1). Professor Lonneke Vervelde, Dr Kate Sutton & Proxima Concepts Ltd. 'Evaluation of a novel adjuvant for mucosal immunistion of poultry'.
Amount £12,830 (GBP)
Funding ID BB/R506564/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 03/2019
 
Title Production of CSF1R-eGFP/mApple transgenic chicken lines that are homozygous for the MHC locus. 
Description Production of CSF1R-eGFP/mApple transgenic chicken lines that are homozygous for the MHC locus. This will allow cell transfer between birds without rejection and will therefore be a major resource for avian immunology researchers. 
Type Of Material Biological samples 
Provided To Others? No  
Impact These birds will be available to other groups once we have a large enough flock. 
 
Title breeding of homozygous transgenic chicken lines 
Description Breeding and characterisation of CSF1R-eGFP/mApple transgenic chicken lines (generated on BBSRC grant BB/H012559/1). In the current grant both CSF1R-eGFP/mApple transgenic chicken lines were bred to produce birds homozygous for the CSF1R-transgene. This means that all experimental chicks produced from these birds will be transgenic. The net effect of this is a significant reduction in the amount of birds bred for experimental purposes. 
Type Of Material Biological samples 
Provided To Others? No  
Impact These birds will be available to other groups once we have a large enough flock. 
 
Description Collaborator for grant BB/M003094/1 
Organisation Semmelweiss University
Department Faculty of Medicine
Country Hungary 
Sector Academic/University 
PI Contribution Exchange of biological samples and reagents
Collaborator Contribution Exchange of biological samples and reagents
Impact exchange of antibodies to detect avian cellular markers
Start Year 2015
 
Description Collaborators on grant BB/M012069 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed joined animal experiment related to the grant objective 1.
Collaborator Contribution Performed joined animal experiment related to the grant objective 1.
Impact No output yet, project started recently.
Start Year 2015
 
Description Collaborators on grant BB/M028305 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany 
Sector Academic/University 
PI Contribution Performed pilot study to investigate susceptibility of chicken lines to APEC.
Collaborator Contribution Developed histological tools.
Impact No outputs yet, project started recently.
Start Year 2015
 
Description Proxima 
Organisation Proxima Concepts Limited
Country United Kingdom 
Sector Private 
PI Contribution Generated preliminary data to apply for funding which was successful.
Collaborator Contribution Supplied material to test in in vivo experiments to apply for funding.
Impact Obtained multiple joined small and large research grants
Start Year 2018
 
Description Proxima Ltd 
Organisation Proxima
Country United Kingdom 
Sector Private 
PI Contribution After exchange of material to study antigen targeting and uptake, our team performed animal experiments and generated preliminary data for further funding applications.
Collaborator Contribution Provided material to study antigen targeting and uptake.
Impact We are currently writing a follow up grant.
Start Year 2017
 
Description British Society of Immunology Annual Conference Liverpool 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I presented a poster at the Annual British Society of Immunology Conference in Liverpool on the 2-5th of December 2019.
My abstract and poster were on display for over 500 attendees and it was a great opportunity to showcase the progress we have made in elucidating macrophages and dendric cells in the chicken spleen. I had a number of attendees come and visit my poster and I got to engage with a number of scientist outwith my field and within the field of mammalian
I was short-listed for poster prize.
Year(s) Of Engagement Activity 2019
URL https://www.immunology.org/events/british-society-for-immunology-congress-2019
 
Description Edinburgh Science Festival 2019 National History Museum of Scotland 
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 I carried out public engagement for the DAN series undertaken by the Roslin Institute to mark the 100 years of genetics at the University of Edinburgh. The engagement included a number of activities to show the public how the Roslin Institute can alter the DNA of animals to help combat disease in pigs, fish, chickens and cattle. There was a number of activities that were aimed for adults and I had some Farmers attend who were very interested in seeing what scientist do for them! Also school children enjoyed the activities which were both fun and educational.
Year(s) Of Engagement Activity 2019
URL https://www.sciencefestival.co.uk/mediaLibrary/other/english/46758.pdf
 
Description Immunology conference (Germany) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Oral presentation at the avian immunology research group meeting in Germany, Sept 2016.
Audience included industry members post graduate students, junior and senior scientists.
UK was voted to be the organisers of the next meeting in 2018 in Oxford and I will be part of the organising team. This will lead to great publicity for the funded avian immunology research funded by the RCUK.
Year(s) Of Engagement Activity 2016
URL http://www.airg2016.vetmed.uni-muenchen.de/airg_2018/index.html
 
Description Invited internal talk-The Roslin Institute Thursday Seminars 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I was invited to present an internal seminar at the Roslin Institute. I presented the progress of my work to colleagues. This was a great opportunity to colleagues to see what area of research i do and my skills have while doing so. I had a number of questions from the audience members. It was an engaging talk.
Year(s) Of Engagement Activity 2019
 
Description Invited lecture at MSD-Animal Health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Invited lecture at MSD-Animal Health in Boxmeer the Netherlands, with WebEx connection to all other location o MSD -AH around the world.
Year(s) Of Engagement Activity 2019
 
Description Invited lecture at the annual BSI meeting 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I gave an invited lecture in the session Immune surveillance and lymphoid tissue architecture at the annual BSI congress in Liverpool. It sparked questions and request fo further info and collaboration.
Year(s) Of Engagement Activity 2019
URL http://www.bsicongress.com/sessionspeakers
 
Description Invited speaker at the fifth Edinburgh Infectious Diseases Annual Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Talk at the fifth Edinburgh Infectious Diseases Annual Symposium, sparked vivid discussions and awareness of the use of livestock animals for fundamental research.
Year(s) Of Engagement Activity 2016
URL http://www.eid.ed.ac.uk/symposium2016
 
Description Invited talk Avian Genetics and Immunology meeting Guilford UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk at a newly organised group of scientist in an attempt to join up geneticist and immunologist to promote future collaborations.
Year(s) Of Engagement Activity 2017
URL https://10times.com/avian-genetics-and-immunity
 
Description Leeds food Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Adam Balic - RI fellow - is a committee member of the Leeds Food Symposium. In that role I have organised a symposium in April 2017 "Birds for the Table". In this public symposium will talk about the development of the modern poultry industry and the role of basic research, including the production of GM chickens, in this process.
Year(s) Of Engagement Activity 2017
URL http://www.leedsfoodsymposium.org.uk/2017_Symposium.html
 
Description Participation in an open day or visit at my research institution - Roslin Open Day by Kate Sutton 
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 I designed a public engagement event with the bio-imaging team at the Roslin Institute to demonstrated the Multi-photon microscope to the public. I designed an activity for visitors to use the microscope and instructed them on the work we do for poultry immunology.
Year(s) Of Engagement Activity 2018
 
Description Press release related to a publication 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Roslin communications team together with the the PI and co-I a story about the latest publication in reation ot this BBSRC funding. The story has also been picked up by Animal Pharm, Poultry World and Feedstuffs. In addtion it was put on the Roslin website and based on the analytics of the Roslin website the news story "Chicken gene find could help improve vaccines" was among the most read news on our website in 2019, with 720 readers. Many others saw it on our and the PIs social media channels including twitter and there have been some compliments to the image we used.
https://animalpharm.agribusinessintelligence.informa.com/AP016579/Breakthrough-Roslin-study-could-instigate-better-poultry-vaccine-design
https://www.feedstuffs.com/nutrition-health/chicken-gene-find-could-help-improve-vaccines
Year(s) Of Engagement Activity 2019
URL https://www.ed.ac.uk/roslin/news-events/latest-news/chicken-gene-find-could-help-improve-vaccines
 
Description Public engagement at 2017 Edinburgh's mini-Maker's Faire 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Roslin Institute had a presences at the 2017 Edinburgh mini-makers faire-this event highlights the diversity of human creativity by bringing together makers from all sorts of unexpected areas under one big roof. This is a chance for the whole family to come out and interact with robotic puppets, 3D printers, model boats, motorised skateboards and science experiments. We held a felting event showcasing the work we do by making felts of common agricultural animals, bacteria and viruses and using it as means to inform people of the work we undertake at the Roslin Institute. It was a great platform to speak to both a young and older generation and informing them of what the Institute undertakes and some of the jobs in the STEM sector.
Year(s) Of Engagement Activity 2017
URL https://www.roslininnovationcentre.com/news/sharing-our-science
 
Description Roslin Open Day by Lonneke Vervelde 
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 Public/other audiences
Results and Impact Around 500 members of the public visited the Easter Bush Campus for our annual Open Day, part of the Midlothian Science Festival, to find out about our research, clinical and teaching work.
Year(s) Of Engagement Activity 2017
URL https://www.ed.ac.uk/roslin/community-engagement/public-events/events-archive/campus-open-day
 
Description Talk/Presentation at the GENIE (Glasgow and Edinburgh Network for Immunology Excellence) 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact I presented an scientific talk on the progress of elucidating the chicken splenic mononuclear phagocytic system in the chicken spleen. a number of audience members asked questions and the attendees were from across the east and west of Scotland. The talk/presence at the conference impacted on collaborations, I had an opportunity to speak with fellow scientist from Glasgow and Edinburgh.
Year(s) Of Engagement Activity 2019
URL https://www.immunology.org/events/eig-and-wsig-joint-symposium-2019
 
Description The Royal Highland Show- The Roslin Institute stand 
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 Public/other audiences
Results and Impact I demonstrated the work the Roslin Institute does to trace and track salmonella outbreaks via deep sequencing and how collecting samples can lead to better surveillance and prevention of outbreaks in the UK.
Year(s) Of Engagement Activity 2018
 
Description interviewed by Sunday Herald In 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Adam Balic interviewed by journalist Judith Duffy for a feature she wrote about transgenic animal research at Roslin "'Dolly' scientists at the forefront of new genetic engineering revolution", 27 November 2016
Year(s) Of Engagement Activity 2016
URL http://www.heraldscotland.com/news/14930660.__39_Dolly__39__scientists_at_the_forefront_of_new_genet...