CSF1 IL34 and the function of Macrophages in Avian Development

Macrophages are large white blood cells that are the first line of defense against pathogens, but also contribute to much of the pathology of infectious and inflammatory disease. Macrophages are also the bodies cellular waste disposal system, and they are needed for wound healing and for many aspects of normal development. Chickens are of interest because they are an economically important livestock species, they are tractable model in which to study development, and they are vectors for diseases that can affect humans including bacteria that cause food poisoning and avian influenza. This project aims to understand how the production of macrophages is controlled in birds and the function of macrophages in embryonic development. Our hypothesis is that two growth factors, macrophage colony-stimulating factor (CSF-1) and interleukin 34 (IL-34) act through a common receptor (the CSF-1 receptor) to promote the production, migration and function of macrophages in an embryo. In turn, the macrophages are needed for the normal process of organ formation and overall growth in the embryo. To address this hypothesis, we propose to make transgenic animals in which all of the macrophages are tagged with a fluorescent transgene so we can monitor when they appear and how they move about in the embryo. We will make the two growth factors as recombinant proteins, and make antibodies that prevent their actions. And finally, we will test the hypothesis by introducing the factors, or antibodies, into the developing embryo in the egg, to see whether macrophage production or location can be altered, and whether this changes the course of normal development. These are experiments that cannot be done easily in mammals, because the embryo cannot be access in the uterus. If our hypothesis is correct, we will identify candidate modulators of chicken immunity and growth, and also gain an insight into normal development that might be relevant to understanding human pregnancy and developmental defects.

Macrophages are key cells involved in innate immunity and homeostasis. This project addresses the question of how their production is controlled, and their function in embryonic development in chickens. The project has two major parts. The first involves the detailed characterisation of a candidate macrophage-specific gene, the macrophage colony-stimulating factor (CSF1R) receptor (CSF1R), in the chicken with the objective of producing a CSF1R-EGFP reporter transgenic chicken line. Successful achievement of this objective will enable detailed study of the generation, location, migration and function of macrophages in embryonic development and in adult birds. A second approach to this objective will involve the generation of yolk sac chimaeras in which the donor expressed EGFP in all cells. The second part of the study involves analysis of the signals that control the production and differentiation of macrophages in the chicken, based upon our identification of our identification of the avian counterparts of the CSF1R ligands, CSF1 and IL-34. We propose to produce the recombinant ligands and receptor, and to make antibodies against them, to determine where they are expressed, and to examine the consequence of increasing, or interfering with their activity during development, taking advantage of the unique access to the embryo in ovo. We will also address the question of whether CSF1 and IL34 bind in distinct modes to the CSF1R, and possibly generate distinct outcomes. This will involve selective mutagenesis of the receptor, and also screening for monoclonal antibodies that can selectively inhibit the actions of one, or other ligand.,

WHO WILL BENEFIT FROM THIS RESEARCH? This project is bridges fundamental basic science and applied science. Scientific Community will benefit from an increased knowledge of the fundamental developmental biology of the innate immune system, based upon the unique features of the chicken system. The Industry and General Public will benefit from improved poultry production. It is envisaged that CSF1 and IL34 could have impacts on both meat and egg production. Poultry are a major livestock industry and source of protein in the UK (850,000,000 birds per annum) and are especially important to the third world. Even small changes in productivity will have very large economic impact. HOW WILL THEY BENEFIT FROM THIS RESEARCH? Scientific community: This project may yield new insights and players involved in the genetic and developmental control of innate immunity and the link between immunity and normal development and growth. Broadening the knowledge base for basic vertebrate immunology research is particularly valuable given the different outcomes in the arms race between hosts and pathogens and the potential for zoonotic infections to develop from birds (especially avian flu). Industry: The project has already produced protected IP, which will be exploited to either (a) produce recombinant proteins for application during chicken production (and by extension, in mammalian and human clinical applications) (b) provide a rationale base for genetic selection of animals that vary in expression of the proposed regulators CSF1 and IL34 Public: improvements in animal health and welfare enabled by the outputs from this project would not only meet the public's aspirations for animal production systems but also potentially improve the sustainability of chicken production systems and thus minimize the cost of chicken products. WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? Scientific community: Publication in the open access refereed scientific literature will be primary means for communicating our findings and hence potential benefits to the scientific community. We will also present our results at scientific meetings, including immunology meetings, i.e. not solely animal genetics meetings, in order that we engage with scientists for whom our approach is novel. Industry: We have a close partnership agreement with Pfizer Animal Health, and existing IP has been appropriately protected. The path for development of the IP is clear, and the proposed research will add value to that IP. Public: We (will) provide information about our research through our web site (with project-specific information), talks and discussion groups and direct interaction with the media. The Roslin Institute encourages clear and open communication and has a policy of promoting Public Engagement by means of interaction with the media, presentations, publications, exhibitions and schools activities. The Institute provides support for staff and students wishing to undertake such activities. The Roslin Institute has a Scientific Administrator who oversees both internal and external communication of the research performed at the institute. Track record: we have an excellent track record for facilitating the benefits of our research as outlined in greater detail in the Impact Plan attached to this proposal.