CSF1R in homeostasis and immunity

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

The sequence of DNA in the genome of a mammal such as a mouse or a human contains a code that specifies the way that a body develops and grows. Specialised types of cells use only parts of the genome to generate their function; for example a red blood cell must produce the proteins (globin) that are needed to carry oxygen, where these proteins are not needed in the liver. This project is concerned with deciphering the code within DNA that ensures that the genes that code for particular products are produced in the right place at the right time. It deals specifically with a model gene, the CSF1R gene, which is needed for the production and function of large white blood cells called macrophages. The only we can dissect the function of individual elements of the code is to remove them from the genome. In this project we will use mouse transgenic technologies to determine what happens to CSF1R is we take out part of the code that controls where and when it is produced. The second part of the project applies the tools generated to determine the function of macrophages. These cells are found in every organ in the body. They are part of the body's defense against infections, but they also contribute to repair and regeneration. Their function is controlled by the CSF1R gene, and we are developing therapies that target that gene. The mouse models will enable us to identify and test new therapies.

Technical Summary

Cellular differentiation requires that particular lineage-specific genes are expressed at the right level, and the right time, coordinated with all of the other genes that are required for a particular cellular function. At the individual locus level, such coordination involves the complex interaction of promoters, enhancers, silencers and boundary elements. There have been few mammalian genes in which each of these elements has been identified and its function studied in detail, so there are no general models of mammalian locus control. The current project aims to identify the control elements of a complex gene, that encoding the CSF-1 receptor. The gene is of particular interest because it controls the development of macrophages, central cells in innate immunity. It is targetted by mutations in neurodegenerative disease, and it is also a target for therapies, both agonists and antagonists. It is also tractable as a model system because we now have genome scale information identifying the boundaries of the transcriptional unit, all of the elements that are in open chromatin in macrophages and their conservation in mammalian genomes, and all of the candidate transcriptional regulators that are expressed in cells that express CSF1R, and the way that their expression correlates with CSF-1 receptor expression in cells undergoing differentiation. The only way that we can now progress our understanding of this locus is to delete the proposed control elements in the germ line. This has become feasible with the advent of CRISPRs. We will generate a range of CSF1R mutants with deletions in each of the putative control elements. In the second phase of the project, we will use those lines to examine the function of CSF1R-dependent macrophages in development, immunity, physiology and homeostasis.

Planned Impact

Who will benefit from our research?
This research has both basic and applied aspects. The scientific community will benefit through the generation of knowledge and models with wide applications. These models also have commercial applications in drug development and academic research on macrophage biology. Macrophages in general, and the CSF1/CSF1R pathway in particular, are of major interest as therapeutic targets. The animal models we generate will be of great utility in preclinical studies, and will also highlight potential side effects of intervention.
How will they benefit from this research?
Through the distribution of knowledge and research tools generated in the project. In particular, the mouse lines will be deposited with the Jackson Labs for free distribution, and reagents such as the CSF1-Fc and Csf1r promoters have been sent to many laboratories around the world.
What will be done to ensure that the benefits from this research arise?
Scientific community: Publication will be the primary means for communicating our findings and hence potential benefits to the scientific community. The research has a wide inter-disciplinary scope. We will also present our results at scientific meetings and will particularly seek to engage a wide spectrum of researchers from the fields of clinical medicine, immunology, veterinary pathology, genetics and genomics and to emphasise the cross-disciplinarity of our research. We will continue to publish in high profile journals, and will also generate images and movies displayed on our macrophage community website, www.macrophages.com (which already has a library of images of the MacGreen transgenic line).
Industry: We already interact with companies in the animal and human health and biotechnology industries, notably Zoetis, Amgen, Roche, Plexxikon, Synpromics, Toughlight in exploring applications of CSF1 and CSFR1 antagonists and the use of our transgenics and vectors. Roslin has two permanent business development staff who have helped to host mutliple events each year specifically showcasing the institute's work to different industry target markets. Our existing mouse transgenic reporter lines have been widely-distributed, and have been used in previous and current drug development partnerships. Although it is outside the direct province of the MRC, CSF1 and CSF1R are also of interest to the animal breeding companies with whom we collaborate.
Public: We provide information about our research through our web sites (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. Roslin provides support for staff and students wishing to undertake such activities. The Roslin Institute's Scientific Administrator oversees both internal and external communication of the research performed at the institute.
Track record: The Roslin Institute has an outstanding track record for translating the outcomes of its research. We hold regular Industry Open Days, and have more than 70 existing industry partnerships.

Publications

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Pridans C (2021) Transcriptomic Analysis of Rat Macrophages in Frontiers in Immunology

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Pridans C (2018) Macrophage colony-stimulating factor increases hepatic macrophage content, liver growth, and lipid accumulation in neonatal rats. in American journal of physiology. Gastrointestinal and liver physiology

 
Description Identifying the role of microglia in mediating ASD-relevant phenotypes in the developing adult brain
Amount £166,244 (GBP)
Organisation Simons Initiative for the Developing Brain 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 03/2021
 
Title Csf1r-FIRE deficient mice 
Description The enhancer for Csf1r (FIRE) was deleted from the mouse germline using CRISPR/Cas9. These mice lack macrophages in the epidermis, heart, kidney, peritoneal cavity and the brain (microglia). They are helthy and fertile and survive to adulthood. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact These mice are the first GM mice that lack microglia (the macrophages of the brain) and survive to adulthood. Other mice that lack microglia (such as the Csf1r deficient mice) do not survive beyond 3 weeks. Previous studies have relied on using CSF1R inhibitors to deplete microglia in order to determine their functions. However, these inhibitors are not specific for CSF1R. So the Csf1r-FIRE mice are a valuable tool for reseachers interested in microglia function and also during development, function and disease states. These mice have resulted in new worldwide collaborations as summarised below. The majority have begun since the publication went public on July 19th, 2019. Edinburgh: 10 Germany: 2 US: 6 Belgium: 2 
URL https://www.ncbi.nlm.nih.gov/pubmed/31324781
 
Title Csf1r-FusionRed mice 
Description We developed a new CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2020 
Provided To Others? Yes  
Impact Mice have been shared with laboratories around the world, resulting in new collaborations 
URL https://pubmed.ncbi.nlm.nih.gov/33139489/
 
Title E631K mutant mice 
Description Mice have a E631K mutation in Csf1r which is found in the human neurodegenerative disease ALSP 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2022 
Provided To Others? Yes  
Impact Publication accepted 5/2/22 
 
Description Cardiac macrophages 
Organisation German Centre for Cardiovascular Research
Country Germany 
Sector Public 
PI Contribution Input to project
Collaborator Contribution Performed experiments
Impact Paper under revision at Nature Cardiovascular Research.
Start Year 2018
 
Description Microglia and myelin 
Organisation University of Edinburgh
Department MRC Centre for Reproductive Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Input to the project
Collaborator Contribution Performed the experiments
Impact Paper in Nature currently under revision
Start Year 2018
 
Description Microglia and prion disease 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Input to project
Collaborator Contribution Performed experiments
Impact Paper under revision at Cell Reports
Start Year 2018
 
Description Role of microglia in AD 
Organisation University of California, Irvine
Country United States 
Sector Academic/University 
PI Contribution Generation of mice with a deletion of the Csf1r enhancer (FIRE)
Collaborator Contribution FIRE deficient mice were crossed to a mouse model of AD to define the role of microglia in disease
Impact Manuscript currently being prepared
Start Year 2017
 
Description Role of microglia in breast > brain cancer metastasis 
Organisation University of California, Irvine
Country United States 
Sector Academic/University 
PI Contribution Generation of FIRE deficient mice
Collaborator Contribution Partners used a breast > brain cancer metastasis model in FIRE deficient mice to examine the role of microglia
Impact Manuscript being prepared
Start Year 2019