Control of macrophage multinucleation in health and disease

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.

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.

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.