Cloning and expression of feline colony stimulating factor receptor (CSF-1R) and analysis of the species specificity of stimulation by colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34).

AbstractColony stimulating factor (CSF-1) and its receptor, CSF-1R, have been previously well studied in humans and rodents to dissect the role they play in development of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, IL-34 has been described in several species. In this study, we have cloned and expressed the feline CSF-1R and examined the responsiveness to CSF-1 and IL-34 from a range of species. The results indicate that pig and human CSF-1 and human IL-34 are equally effective in cats, where both mouse CSF-1 and IL-34 are significantly less active. Recombinant human CSF-1 can be used to generate populations of feline bone marrow and monocyte derived macrophages that can be used to further dissect macrophage-specific gene expression in this species, and to compare it to data derived from mouse, human and pig. These results set the scene for therapeutic use of CSF-1 and IL-34 in cats

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background: The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity

[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor (CSF1) receptor

BACKGROUND: Macrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model. RESULTS: Based upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP(+) donors. In both cases, the transferred cells gave rise to large numbers of EGFP(+) tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP(+) cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings. CONCLUSIONS: The data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0121-9) contains supplementary material, which is available to authorized users

Characterisation of the chicken mononuclear phagocyte system

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

Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products

12 páginas, 5 figuras.-- et al.Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF-1 via the activation of the CSF1R. A recently discovered cytokine, IL-34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF-1, IL-34, 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 is expressed in the developing chick embryo in a pattern consistent with macrophage-specific expression. Chicken CSF-1 and IL-34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co-evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates.This work was supported by grant BB/D010705/1 and BBSRC Institute Strategic Program grant to The Roslin Institute.Peer reviewe

Production and characterisation of a monoclonal antibody that recognises the chicken CSF1 receptor and confirms that expression is restricted to macrophage-lineage cells

Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology

Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products

Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF-1 via the activation of the CSF1R. A recently discovered cytokine, IL-34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF-1, IL-34, 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 is expressed in the developing chick embryo in a pattern consistent with macrophage-specific expression. Chicken CSF-1 and IL-34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co-evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates

HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA

The mammalian innate immune system is activated by foreign nucleic acids. Detection of double-stranded DNA (dsDNA) in the cytoplasm triggers characteristic antiviral responses and macrophage cell death. Cytoplasmic dsDNA rapidly activated caspase 3 and caspase 1 in bone marrow-derived macrophages. We identified the HIN-200 family member and candidate lupus susceptibility factor, p202, as a dsDNA binding protein that bound stably and rapidly to transfected DNA. Knockdown studies showed p202 to be an inhibitor of DNA-induced caspase activation. Conversely, the related pyrin domain-containing HIN-200 factor, AIM2 (p210), was required for caspase activation by cytoplasmic dsDNA. This work indicates that HIN-200 proteins can act as pattern recognition receptors mediating responses to cytoplasmic dsDNA.</p

HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA

The mammalian innate immune system is activated by foreign nucleic acids. Detection of double-stranded DNA (dsDNA) in the cytoplasm triggers characteristic antiviral responses and macrophage cell death. Cytoplasmic dsDNA rapidly activated caspase 3 and caspase 1 in bone marrow–derived macrophages. We identified the HIN-200 family member and candidate lupus susceptibility factor, p202, as a dsDNA binding protein that bound stably and rapidly to transfected DNA. Knockdown studies showed p202 to be an inhibitor of DNA-induced caspase activation. Conversely, the related pyrin domain–containing HIN-200 factor, AIM2 (p210), was required for caspase activation by cytoplasmic dsDNA. This work indicates that HIN-200 proteins can act as pattern recognition receptors mediating responses to cytoplasmic dsDNA