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

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.

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.

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.