Characterisation of the chicken mononuclear phagocyte system

Abstract

Macrophages are present in every tissue, and have a central role in immune responses, development and homeostasis. Typically recognised as scavenger cells phagocytising pathogens and dead cells, macrophages also regulate the innate and adaptive immune responses via the secretion of cytokines. In mammals, the differentiation, proliferation, and survival of macrophages are controlled by macrophage colony-stimulating factor, or CSF1, which acts through the CSF1 receptor (CSF1R), a ligand-dependent protein tyrosine kinase. IL34, a more recently discovered cytokine with a differential expression, shares the CSF1R. Natural or artificial knock out of these genes in mice and rats depletes macrophage populations with consequent pleiotropic effects on development of multiple organs. The mammalian CSF1R is exclusively expressed on the cells of the macrophage lineage, and their progenitors. For this reason, the CSF1R promoter has been used to generate fluorescent reporter transgenic mice, to permit analysis of macrophage function in vivo. Macrophages are present in very large numbers from midgestation in mice, but dynamic studies of their biology are difficult in a mammal. The chick has been used extensively as a developmental biology model because of the ease of visualisation and manipulation in ovo. It has the added advantage of being economically important. At the start of this project, the factors that control avian myelopoiesis had not been identified. Indeed, CSF1 was not identified in the chicken genome. The primary objective of this research was to characterise the chicken mononuclear phagocyte system. To this end, the CSF1, IL34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage-specific promoters and enhancers, and the CSF1R gene was shown to be expressed specifically in macrophages of the developing chick embryo. These observations formed the basis of the generation of a chicken CSF1R reporter transgenic by a colleague in the laboratory. Structure-based modelling, comparative amino acid sequence analysis and co-evolution study across all vertebrates demonstrated the conservation of the IL34/CSF1/CSF1R complex in birds. Modelling also suggested that IL34 was a four helix bundle factor, structurally related to CSF1, which was subsequently confirmed by published crystal structure. To show that these factors were active in birds, chicken CSF1 and IL34 were expressed in HEK293 cells. Although chicken CSF1 lacked the interchain disulphide present in the mammalian protein, it formed a dimer. Both factors were able to promote the generation of pure macrophage cultures when added to chicken bone marrow. The specificity of action of chCSF1 and chIL34 on chCSF1R was assessed using murine myeloid IL-3 dependent Ba/F3 cells stably transfected with chCSF1R. Either chCSF1 or chIL34 alone could substitute for IL3 in receptor-expressing cells and caused them to differentiate further into the monocytic lineage pathway and to undergo growth arrest. The avian factors were not active on mammalian CSF1R. The observed species specificity and inactivity of the CSF1R inhibitor GW2580 in chicken were linked to the dissimilarities between the avian and mammalian CSF1/IL34/CSF1R proteins. To enable functional studies in vivo, a project was initiated to produce a monoclonal antibody against chicken CSF1R. Binding of the monoclonal to cells demonstrated that CSF1R was, indeed, monocyte-macrophage restricted. Chicken CSF1 was expressed as a fusion protein with the domains 3 and 4 of the chicken immunoglobulin. This increased the half-life of the recombinant chCSF1 without impairing its activity. Injection of chCSF1-Fc in the neural tube of stage HH21 chick embryos stimulated the proliferation of embryonic macrophages. Similarly, four consecutive daily injections of chCSF1-Fc in chicken hatchlings resulted in an increase in tissue macrophage number, notably in the spleen, liver and lung. To investigate the pathway of development of macrophages during embryogenesis, bone marrow from chicken ubiquitously expressing EGFP was transplanted into the circulation of stage HH16-17 embryos. The results demonstrated effective colonisation of the hematopoietic organs, and highlighted the presence of large numbers of macrophages in embryonic tissues, similar to those seen in MacGreen mice. The results are discussed in the context of the proposed yolk sac origin of some macrophage subpopulations, such as microglia cells and Langerhans cells, and the presence of a clonogenic macrophage-committed progenitor in the bone marrow that is distinct from the pluripotent stem cell. Bone marrow-derived macrophages (BMDMs) grown in CSF1 have been used extensively as a model to understand gene regulation in mice. The cloning and expression of chicken CSF1 permitted the production of large numbers of BMDMs from chicken bone marrow. To enable the characterisation of chicken macrophages and comparison to mammalian BMDMs, the gene expression profile of these cells was examined using RNAseq. For comparison, mid incubation embryos and a fibroblast line were also profiled. These data could identify several novel chicken macrophage-specific transcripts that may assist in further dissection of macrophage differentiation in birds and contribute to chicken genome annotation. Overall, this project has demonstrated that the CSF1/IL34/CSF1R system is conserved in birds, and controls the generation of monocytes and tissue macrophages. It has provided the tools to enable detailed analysis of the function of this system in embryogenesis and immunity.