Control of macrophage multinucleation in health and disease

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

The proposed project specifically addresses the MRC Strategic Plan in the areas of "tissue disease and degeneration" and "molecular datasets and disease". Macrophages are cells of the immune system that can fuse to form giant cells with multiple nuclei in peripheral tissues and specialist bone-resorbing osteoclasts in the skeleton. The fundamental physiological process of multinucleation is poorly understood, but is essential for the normal immune response and regulation of bone mass. Granulomas containing multinucleated giant cells are classically seen in chronic inflammatory disorders and infectious diseases including Crohn's disease, sarcoidosis and tuberculosis, whilst increased osteoclastic bone resorption causes osteoporosis and underlies cancer-induced bone loss. We propose that genes driving macrophage multinucleation also control signalling pathways that are essential for normal bone turnover and the inflammatory response, but which are dysregulated in osteoporosis and chronic granulomatous disease.

In this project we will investigate the molecular mechanisms controlling macrophage multinucleation in health and disease. We have previously described a network of 134 genes involved in macrophage multinucleation (MMnet), and will now identify the ten genes with the largest effect on macrophage multinucleation using a cell culture-based screening approach. We will next establish their physiological importance to bone and the immune system by analysing the effects of universal gene deletion. Two genes will be selected on the basis of their novelty, effect size, function, experimental tractability and relevance to human disease for detailed studies in which the genes will be deleted specifically in macrophages. Studies using established disease provocation models will then determine whether deletion of the two genes in macrophages protects against development of osteoporosis and chronic inflammatory disease. These studies will identify signalling pathways that play a fundamental role in the development and progression of granulomatous disease and provide a new understanding of macrophage biology in health and disease.

Technical Summary

We hypothesise that key genetic determinants of macrophage multinucleation orchestrate fundamental signalling pathways that regulate bone resorption and are perturbed in granulomatous disease. We will use genome-wide expression and systems-genetics, rapid-throughput phenotyping, and disease provocation models in order to:

1. Undertake an RNAi screen and RNAseq in primary macrophages to identify key genetic determinants and pathways regulating macrophage multinucleation

2. Identify 10 genes with the largest inhibitory effect on macrophage multinucleation, and establish their physiological importance using skeletal samples from corresponding knockout models obtained from the IKMC

3. Select two genes on the basis of effect size, tractability, function and translation to human disease and investigate the molecular mechanisms of macrophage multinucleation

4. Generate monocyte-macrophage specific knockout lines for the two selected genes, perform detailed skeletal phenotyping and use provocation models for osteoporosis, inflammatory arthritis and glomerulonephritis

We will use siRNA knockdown for each of 134 genes that comprise a network controlling macrophage multinucleation. We will identify 10 genes with the largest effect and perform RNAseq and systems-level bioinformatics to determine the key signalling pathways essential for multinucleation. Skeletal consequences of global deletion of these 10 genes will be determined by X-ray microradiography, micro-CT and biomechanical testing. Analysis of developmental and adult phenotypes will be performed in two macrophage specific knockout lines using additional methods including electron microscopy, histomorphometry and primary cultured osteoblasts and osteoclasts. Ovariectomy, collagen-induced arthritis and macrophage-dependent crescentic glomerulonephritis disease models will be used to determine the effect of gene deletion on susceptibility to osteoporosis and chronic inflammatory joint and kidney disease.

Planned Impact

The proposed studies will lead to a new understanding of the fundamental process of cell fusion and multinucleation, and its role in health and disease. This research will have broad impact with beneficiaries in the scientific, clinical and wider public domains. Some benefits will be realised within the duration of the project: these include advances in understanding of basic cell biology and physiology together with development of novel methodologies. The full translational potential of this research to the clinical area will inevitably be realised over a longer timeframe, whilst public understanding and outreach will continue throughout the project and beyond.

WHO WILL BENEFIT FROM THE RESEARCH?
1. Scientific researchers in the fields of basic cell biology, mouse and human genetics, bioinformatics, imaging, bone biology, inflammation and infection.

2. Clinical scientists and clinicians working in the following specialties: rheumatology, infectious diseases, nephrology, respiratory disease, gastroenterology, hepatology, metabolic bone disease, care of the elderly and oncology, together with other health care providers including general practitioners.

3. Patient groups and disease-related charities including organisations supporting patients and carers with Crohn's disease, Wegener's granulomatosis, sarcoidosis, systemic lupus erythematosus, osteoporosis, myeloma and other osteolytic malignancies. Examples include the Crohn's and Colitis UK, Vasculitis UK, Sarcoidosis and Interstitial Lung Association, Lupus UK, National Osteoporosis Society and Myeloma UK.

4. Ethnic minorities at particular risk of tuberculosis including communities from sub-Saharan Africa, Southeast Asia including India, Pakistan, Indonesia and Bangladesh, Russia, China, South America and the Western Pacific region including Vietnam and Cambodia.

5. Pharmaceutical industry

HOW WILL THEY BENEFIT FROM THIS RESEARCH?
1. Advancements in understanding of diverse fields of basic and clinical science including in physiological and pathophysiological processes.

2. Education of scientific researchers, clinical scientists, clinicians, patient support groups, patients and the wider general public by dissemination of research findings and broader information.

3. Training of research scientists, clinicians and students in new advances in basic cell biology, mouse and human genetics, high-throughput screening methodology, bioinformatics, imaging, bone biology and the fields of inflammation and infection.

4. Identification and development of novel biomarkers and potential drug targets for the diagnosis, monitoring and treatment of chronic inflammatory, infectious and skeletal diseases.

Publications

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Moreno-Moral A (2017) Systems Genetics as a Tool to Identify Master Genetic Regulators in Complex Disease. in Methods in molecular biology (Clifton, N.J.)

publication icon
Moreno-Moral A (2017) Systems Genetics as a Tool to Identify Master Genetic Regulators in Complex Disease. in Methods in molecular biology (Clifton, N.J.)

 
Description Training of BSc student (Research)
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Training of pathologist
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Title Gene silencing and measurement of multinucleation in human osteoclasts: A quantitative approach 
Description The first 4 months of the grant was important in establishing an assay to measure quantitatively macrophage multinucleation in humans. We have established a reliable and robust assay to silence genes regulating macrophage multinucleation and measure their effect on cell fusion. 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact This tool is essential to complement our in vivo approaches focusing on bone phenotypes. 
 
Title Single cell RNA-sequencing 
Description Single cell RNA-sequencing (ScRNA-seq) was optimised succesfully on the 10X Genomics platform using peripheral blood cells from a healthy donor 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The use of scRNA-seq will have a considerable impact for all the research projects. Our preliminary results showed that the technique can allow clustering of a heterogenous cell population based on their transcriptome 
 
Title Sorting and purification of human osteoclasts according to their number of nuclei 
Description Osteoclasts are multi-nucleated cells and the transcriptional and epigenetic modifications that derive from multi-nucleation and their effect on cell activity are not well-established. In this grant, we have purified human osteoclasts by FACS-sorting according to their number of nuclei. We isolated mono-nuclear, 2-3 nuclei containing cells and >4 nuclei cells with >90% purity. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2018 
Provided To Others? No  
Impact RNA-sequencing of the 3 cell populations described above revealed fundemantal differences in cell transcriptome that result from multi-nucleation. This method provided results that we currenty pursue for an upcoming publication in this field. 
 
Title orthogonal organic phase separation (OOPS) 
Description unbiased detection of cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA. (TRIZOL-based methodology) 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact N/A 
 
Title Human osteoclast proteomics 
Description Human RANKL-differentiated osteoclast or non-differentiated macrophage proteomics from healthy individuals 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact Preliminary findings likely to lead to a publication 
 
Title Lipidomics profiling in healthy macrophages 
Description Lipid quantification (>200 lipid species) during a TLR4 activation timecourse 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact This is a preliminary dataset and an ongoing collaboration 
 
Title RNA-seq in human osteoclasts according to their nuclei number 
Description RNA-seq dataset from human osteoclasts displaying 1 or 2-3 or >4 nuclei 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact N/A 
 
Title Scleroderma macrophage dataset 
Description RNA-sequencing from scleroderma patient and healthy individual macrophages. The cells were differentiated from the peripheral blood monocytes. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact A recent publication in Annals of the Rheumatic Diseases where the Pi is the corresponding author (PMID: 29348297) 
 
Title time-course of RNA-sequencing in human macrophages 
Description RNA-seq from human macrophages stimulated with LPS (30min, 3h, 8h and 16h) or left unstimulated (basal) 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact N/A 
 
Description Collaboration on NOD2-mediated granulomatous disease 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution We have shared some protocols and data on human osteoclast isolation, multinucleation and gene targetting
Collaborator Contribution Our collaborators in Leuven will provide multinucleated giant cells (MGCs) from patients who have Blau syndrome (NOD2 mutations).
Impact This collaboration has just started.
Start Year 2019
 
Description Collaboration with Siamon Gordon 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Siamon Gordon is one of the most senior researchers in the field of macrophage biology. His research focused on macrophage heterogeneity, differentiation and activation, during development, infection and metabolic disease. His interest in cell fusion lead to the development of a range of monoclonal antibodies to study macrophages in tissues such as bone marrow, spleen and the nervous system. The functional significance of macrophage receptors and giant cell formation remained an interest to the present. As Emeritus he has been immersed in history of macrophage research, from Metchnikoff to the discovery of Dendritic cells by Steinman and Cohn. The collaboration with Siamon Gordon is strictly based on sharing knowledge on macrophage fusion in health and disease.
Collaborator Contribution Please see above.
Impact A review ion macrophage multinucleation is currently under preparation with Siamon Gordon's input.
Start Year 2016
 
Title ABBA (A Bayesian Approach for Analysis of Whole-Genome Bisulfite Sequencing Data) 
Description Allows the detection of differentially methylated regions (DMRs) from whole-genome bisulfite sequencing (WGBS). ABBA is implemented as a Perl/R program, which is available with instructions for download at http://abba.systems-genetics.net/ or via http://www.mrc-bsu.cam.ac.uk/software/bioinformatics-and-statistical-genomics/. 
Type Of Technology Webtool/Application 
Year Produced 2017 
Impact Data sharing with the larger academic beneficiaries; a publication in the journal Genetics (PMID:28213474) 
URL http://abba.systems-genetics.net/
 
Description Imperial College Press Release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact An Imperial College Press Release presented the results published in Nature Communications. http://www.imperial.ac.uk/news/180507/ This resulted in public awareness (some online comments allowed the PI to discuss and communicate the findings with a lay audience).
Year(s) Of Engagement Activity 2017
URL http://www.imperial.ac.uk/news/180507/
 
Description Scleroderma paper press release at Duke_NUS, Singapore 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This is a press release on the latest research published in Annals of the Rheumatic Diseases. The publication had an Altmetric Score of 71 and reached 8 news outlets.
Year(s) Of Engagement Activity 2017
URL https://www.duke-nus.edu.sg/news/genomics-reveals-key-macrophages%E2%80%99-involvement-systemic-scle...