Exploitation of new technologies to advance understanding of avian dendritic cell biology

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) present antigen in birds. We need to gain more information on these processes and we have developed a new tools for identifying and studying these cells and have recently identified a very important type of mononuclear phagocyte which are known as conventional or classic dendritic cells. This is very important as this means that we can use these tools to fully study these cells. The main objectives of this research proposal are to (1) identify and functionally characterise chicken conventional dendritic cells in birds at different ages and in different tissues (2) produce gene edited birds that lack these conventional dendritic cells in order to isolate their specific functions (3) Use the gene edited birds generated in objective 1 to test how important they are to resistance against the bacterial pathogen Salmonella. In the longer term this knowledge help us develop 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 presentation will underpin this. We have developed tools that allow us to identify and manipulate Flt3+ conventional dendritic cells (cDC) in the chicken. Using these tools and exploiting cutting edge gene editing technology to we will build upon this work to further define the biology and function of these cells during normal development and during pathogen challenge.
Objective 1: To determine the ontogeny of Flt3+ cDC in chickens and the manipulation of the development of Flt3+ cells in vivo with recombinant Flt3l-Fc and CSF1-Fc. Determination of when cDC first appear in the developing chicken embryo/post-hatch chick and what organs. Test the potential of a recombinant ligand for Flt3 (Flt3l-Fc) to alter cDC development and function in the chicken; and test the potential of Flt3l-Fc to expand cDC numbers in vivo.

Objective 2: To knockout of the Flt3 gene specifically in CSF1R-expressing cells. Production of two novel parental lines of gene edited chickens and crossing these two lines of birds to produce a birds in which transcription of the Flt3 gene, and hence Flt3 protein production, is specifically terminated in CSF1R expressing cells only. This will produce birds that are deficient in cDC, but not macrophages or monocytes.

Objective 3: 3. To assess the effect of Flt3 lineage restricted knockout on the development of vaccine and non-vaccine induced immunity to the pathogen Salmonella Typhimurium. Once the CSF1R conditional Flt3 knockouts have been generated in Objective 1, we will assess the contribution of the Flt3+ cDC lineage to the development of both vaccine induced and non-vaccine induced immunity to Salmonella Typhimurium.

Planned Impact

This research programme has direct relevance to the strategic priorities of the BBSRC - Animal Health. Infectious diseases are a significant threat to the poultry industry through losses or reduction in production, decreases in egg production/quality, and effects on animal welfare. Vaccination is used to control the major diseases of poultry but the specific cell types 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 immune system. Furthermore, the relatively young age that commercial broiler chickens are vaccinated (embryonic and pre-adult stages) means that current immunological models, which are based largely based on data from adult mammalian studies, may not be relevant. Thus the ability to combat infectious diseases which reduce the health and welfare of in poultry requires not only the development of more efficient vaccines, reagents and functional assays to assess immune function, but also an increased understanding of the immune cell type(s) that interface with disease causing pathogens at relevant developmental stages and tissue niches.

Outputs of the work proposed will include the developmental and phenotypic characterisation of the conventional/classic dendritic cell subset in the chicken in embryonic and pre-adult stage chickens and the generation of a gene edited chicken model which will allow us to distinguish the relative contribution of this cell type to the generation of vaccine and pathogen driven immune responses in comparison to other similar cells type, such as macrophages and monocytes.

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 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 This project aims to understand the biology of chicken conventional dendritic cells (cDC). These cells are hypothesised to be critical components that are required for generation of protective immune responses to pathogens. We have produced specific tools to identify these cells and are producing gene edited chickens in which these cells are specifically absent in order to determine the function of these cells in chickens. We have uncovered a large amount of basic biology on these cells, much of which will have direct consequences for how we think about producing more healthy, immunologically robust chickens. We have found that unlike mammals, chickens only have a single cDC population and that this cell population appears to be specialised for anti-viral responses.
Exploitation Route The finding we have made will have direct application for the development of vaccines to avian pathogens and will inform the breeding of chickens commercially.
Sectors Agriculture, Food and Drink

 
Description BBSRC - Responsive mode research grants
Amount £590,000 (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 BBSRC - Responsive mode research grants
Amount £590,000 (GBP)
Funding ID BB/R003653/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 10/2020
 
Title Antibody - Tim4 antibody 
Description A monoclonal antibody directed against chicken TIM4, a receptor for phosphatidylserine. Phosphatidylserine is expressed on the surface of apoptotic cells. Therefore this monoclonal antibody stains cells that interact with apoptotic cells. 
Type Of Material Antibody 
Year Produced 2019 
Provided To Others? Yes  
Impact In liver, highly-phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of DC-like cells. 
URL http://www.jimmunol.org/content/early/2019/01/08/jimmunol.1800504
 
Title Flt3 antibody 
Description A monoclonal antibody against chicken Flt3. Flt3 is the receptor for the major dendritic cell growth factor Flt3 ligand. 
Type Of Material Antibody 
Year Produced 2017 
Provided To Others? No  
Impact Publication near completion. Antibody will be made available to other researchers upon publication 
 
Title In vitro testing of Flt3 function 
Description We developed an in vitro FLT3L-Fc binding assay that is a direct functional readout of FLT3 ligand binding.. Briefly, wild type and a mutated forms of chicken FLT3 were expressed in murine Ba/F3 cells. Fluorescent labelled FLT3L-Fc was added to these cells and binding was assessed by flow-cytometry. This assay allows us to determine effect of specific mutations on the Flt3 receptor ligand interactions. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact This assay allows us to test function of variations of FLT3 that exist within the chicken population. Using this assay we have identified a deleterious mutation that exists in both inbred experimental birds and native chickens from Africa. Prior to the development of this assay it would not be possible to identify these deleterious mutations without experimentally testing hundreds, if not thousands for birds. 
 
Description Ontogeny of chicken CSF1R-transgene reporter cells. 
Organisation Semmelweiss University
Country Hungary 
Sector Academic/University 
PI Contribution We are actively collaborating on determining the ontogeny of chicken CSF1R-transgene reporter cells. We have produced the CSF1R-transgene reporter line of chickens and have used these to identify and characterise multiple cell types. We have also supplied tissues samples from these lines for further analysis
Collaborator Contribution Experimental procedure, such as embryonic grafting to generate primary data.
Impact Paper published in 2018.. J Anat. 2018 Oct;233(4):401-410. doi: 10.1111/joa.12863.
Start Year 2017
 
Description What makes a microglia? Comparing relatives in the molecular age. 
Organisation Heinrich Heine University Düsseldorf
Department Institute of Neuropathology
Country Germany 
Sector Academic/University 
PI Contribution Our group produced purified chicken brain microglia from macrophage reporter chickens and collected brain tissue for histological stainings, morphological 3D reconstruction.
Collaborator Contribution they examined the the evolutionary divergence of microglia for clues about the ancient and diverse phylogenetic origins of microglial functions by examining 19 different species (including chickens), ranging from leeches to humans. To do this they used histological stainings, morphological 3D reconstruction and sorted microglia based on evolutionary shared microglial markers for next generation single cell sequencing.
Impact Workshop Presentation: Keystone symposia Myeloid cells 2018. Manuscript submitted to Cell in 2019. Geirsdottir et al. Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program. Cell. 2019 Dec. 179(7):1609-1622.
Start Year 2017
 
Description The Avian Immunology Research Group (AIRG) 2018 meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation "Identification and characterisation of avian conventional dendritic cells"
Year(s) Of Engagement Activity 2018