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Exploring Functional and Developmental Osteoclast Heterogeneity in Health and Disease

Final Report Summary - EUROCLAST (Exploring Functional and Developmental Osteoclast Heterogeneity in Health and Disease)

Euroclast was a research and training program focusing on osteoclast biology in the context of osteoclast-driven diseases. Osteoclasts are complex, multinucleated cells that have the unique capacity to degrade bone. Decreased activity of osteoclasts results in high bone density, such as is seen in a group of rare genetic conditions collectively known as osteopetroses. Study of the genetic origin of these conditions has helped to identify several key regulators of osteoclast activity and has helped to devise rational new treatments for osteoclast-driven bone disease, some of which are now entering clinical practise. By contrast, a range of common conditions such as osteoporosis, periodontitis, or rheumatoid arthritis (RA) are characterized by an increased activity of osteoclasts, manifesting in bone loss. The incidence of these chronic conditions is increasing in an ageing –EU- population. Considering that new treatments for osteoclast-driven bone diseases require thorough understanding of osteoclast cell biology, the Euroclast project had two overarching scientific aims. Firstly, it sought to uncover new basic knowledge about the cellular function and regulation of osteoclasts and secondly, it explored whether the differences observed in gene expression in osteoclasts at different bone sites are the result of heterogeneity in this cell population.

Goals and objectives
The multi-site initial training network (ITN) Euroclast brought together 7 leading academic groups and 2 SMEs from 6 EU member states. Together they provided a platform for training of early stage researchers (ESRs). An important goal of the network was to provide excellent training, not only in osteoclast-related techniques, but more broadly, in cell biology and a wide range of generic, research-related topics. These training sessions were delivered by the partners of Euroclast and by the associated partner, who provided training in patenting and knowledge exchange. The training programme was complemented by external trainers in topics such as academic writing, public engagement, working with social media, working in industry, CV writing and interview skills. All ESRs spent some time in the laboratory of one or more of the other partners and all took part in a specifically developed hands-on course held at one of the SME partners, to learn how a new product has to navigate the route to market. To help evaluate the project we invited an osteoclast expert from outside the consortium to each of our consortium meetings. Jointly with the Euroclast partners, these trainers and experts helped equip the Euroclast ESRs, through thorough training and well thought-out research projects, for a career in (bone) cell biology either in academia or in industry. They also provided the ESRs with a wide network of academic and industrial experts at the start of their research careers. All participants attended national and international conferences where they presented the results of the Euroclast projects and various partners had the opportunity to disseminate the findings in training courses and other educational events. ESRs and partners took part in outreach activities such as European Researcher’s Night and local events at their specific Institutions. The Euroclast consortium contributed to the consortium summits of the European Calcified Tissue Society in 2016 and 2017 where European collaborations in the field of mineralised tissues were being showcased to raise awareness of projects and outcomes.

The main questions that Euroclast aimed to address were organised into 4 research work packages (WPs). Studies in WP1 focussed on identification and characterisation of different populations of osteoclast progenitors. WP2 involved studies on the resorption machinery of osteoclasts. Studies in WP3 looked at the regulation of osteoclasts, including in different subsets of osteoclasts and in cell lines. WP4 studied biomarkers for bone/cartilage resorption and the role of vitamin A in bone metabolism. In addition to these scientific WPs, two others, WP 5 and 6, were concerned with dissemination and exploitation, and with management respectively.

Some key outcomes of the studies are given below.
1. There is clear evidence of heterogeneity in osteoclasts.
Under identical conditions, subsets of osteoclast precursors isolated by flow cytometry from mouse or human blood or bone marrow generate different numbers of osteoclasts of different sizes and resorptive capacity that respond differently to osteoclastogenic cytokines such as IL17A, IL-1β and TNFα. We conclude that better knowledge of osteoclast precursor subsets will help to understand pathological resorption in clinical contexts.
2. New methodologies were developed and used to study components of the resorption machinery of osteoclasts.
Live osteoclast imaging using a variety of fluorescent labels, or expressed fluorescently labelled proteins, allowed studies of ruffled border formation in real time. TEM tomography gave highly detailed information about the formation of the ruffled border and vesicular trafficking leading to new hypotheses about enzyme and acid release. Proteomics of osteoclasts and viral knockdown of genes in osteoclast cultures allowed identification and investigation of proteins not previously studied in the context of osteoclastic resorption.
3. Studies in mouse models in vivo and osteoclast cultures ex vivo identified roles of new proteins and pathways in osteoclast resorption.
We found evidence for a role of canonical and non-canonical autophagy in osteoclast resorption. AraP-1 is cricially important for assembly and disassembly of the podosome belt in the sealing zone of osteoclast. PLEKHM1 plays an important role in the positioning of acidic vesicles in the ruffled border area. AP-3 regulates trafficking of lysosomal membrane proteins to the ruffled border. These findings underscore how bone resorption is critically dependent on the correct localisation of vesicles with acidic and proteolytic content and trafficking of membrane proteins, processes that require further studies in the future.
4. We developed new osteoclast cell lines, and new tests for osteoclast activity.
We generated a novel cell line ERHoxb8, with a cathepsin K promoter-driven luciferase reporter, which can be differentiated into osteoclasts and responds to pro-inflammatory cytokines by generating more osteoclasts. This cell line has proved useful for testing the osteoclastogenic effect of sera of patients with inflammatory joint disease. We developed a new ELISA to measure TRAP5a and TRAP5b respectively in human serum to assess bone resorption. By studying the enzymatic requirement for osteoclastic resorption of calcified versus non calcified matrices we showed that osteoclasts use different enzymes on each and hence possess plasticity in their functionality. These findings have found clinical application and add to notion that further understanding of osteoclast function and heterogeneity is required to pharmacologically target pathological osteoclast function.

In additiion, a large number of genetic mouse models was investigated in vivo and ex vivo to investigate the effect of deletion of specific genes with suspected roles in bone resorption. Rescue experiments were performed to heal osteopetrotic osteoclasts. The varied effects of vitamin A on bone in different sites was studied and gene regulation in osteoclast cell lines and subclones investigated. Comprehensive results are reported in the PhD theses of the ESRs and key findings as per above have been reported in publications with more papers in the pipeline.

In conclusion
Our studies have contributed new knowledge on osteoclast heterogeneity and osteoclast function and results continue to be prepared for publication in papers, conference proceedings and PhD theses. So far 5 ESRs have completed their PhD and have found appropriate employment elsewhere (3 work as postdocs in well-established institutions, 1 is managing an academic imaging facility and 1 is working with a pharma company), and all others are close to completion. The ESRs and partners continue to network and collaborate and new collaborations have been initiated as a result of inviting external osteoclast experts to our consortium meetings. Building on the knowledge gained about osteoclast heterogeneity, a new consortium has been formed to explore osteoclasts in the context of inflammatory diseases (IMMUNOCLAST, application under consideration). Euroclast partners have recently been invited to osteoclast workshops to discuss their new findings in relation to rare osteoclast diseases. This satisfies our main goal in terms of training and networking.

Website: www.euroclast.eu