Title: Neutrophils in Bone Ageing and Fracture Repair: Friend of Foe?
Funder: Dunhill Medical Trust

Abstract: Bone is a highly dynamic and metabolically active tissue. Synergy between multiple cell types is required to maintain and repair bone throughout life. However, bone homeostasis is often lost with age, resulting in osteoporosis. Osteoporosis is characterised by reduced bone synthesis in relation to bone resorption, leading to decreased bone mineral density (BMD), increased bone fragility and high fracture incidence. Age is a major predictor of osteoporosis, as is long-term glucocorticoid use. Currently, 3 million people in the UK live with osteoporosis, accounting for half a million fragility fractures annually. The impacts of fragility fractures are significant, often causing long-term pain, debilitation and social isolation. Fragility fractures also carry a substantial economic burden, costing the UK £4.4 billion annually, with costs set to rise by 30% by 2030. Fragility fractures in older people are difficult to treat, in part due to their complex aetiology and age-related changes to the immune system (inflammaging). Further research to characterise age related changes to fracture repair at the cellular and molecular level is required to reduce the morbidity and excess mortality associated with fragility fracture. Inflammaging is characterised by systemic low-grade activation of the immune system and is associated with BMD loss and increased fracture risk. Neutrophils, the most abundant leukocyte, and are the first cells to respond to fracture. Whilst they prevent infection and trigger repair processes after bone trauma, excessive neutrophilic inflammation is negatively associated with fracture healing. Much research has investigated the negative impact of neutrophils on fracture, yet few studies have focussed on the pro-reparative properties of neutrophils in any tissue, including bone. Recently, neutrophils were shown to secrete a fibronectin rich scaffold of extracellular matrix (ECM) in human fractures, proposed to stabilise bone. However, no further studies have attempted to characterise the pro-reparative role of neutrophils in fracture. Neutrophil function is affected by ageing and glucocorticoid use. We hypothesise that: neutrophils contribute towards fracture healing by secreting extracellular matrix in the early phase of repair and that this process is perturbed by ageing. In this project, we will characterise the pro-reparative role of neutrophils in healthy fracture repair using zebrafish as a model, complimented by human neutrophil experiments in vitro to increase translational impact. We will also study the influence of ageing on the neutrophil response to fracture and subsequent impact on bone repair. Zebrafish are established models for studying skeletal biology and disease, due to a high degree of genetic conservation with humans. Crucially, adult zebrafish have translucent fins which allow for dynamic live imaging of cell behaviour during fracture repair. We have published an induced fracture assay, which allows dynamic imaging of the neutrophil response to fracture. We will use this method in combination with transgenic lines, immunohistochemistry and proteomics in young and aged zebrafish to address our hypothesis. Skeletal homeostasis is perturbed by ageing in zebrafish; fins accumulate bone calluses resulting from spontaneous fracture with increasing age. Preliminary data shows laminin and fibronectin within neutrophils recruited to fracture, and that repair is compromised by modulating neutrophils via CXCR2 inhibition. We have also shown that stimulation of human neutrophils in vitro results in expression of ECM proteins. We will deplete neutrophils via 2 methods: nitroreductase-mediated ablation and CXCR2 inhibition, to study the effect of reduced neutrophil activity on bone repair. We will perform fractures in aged zebrafish and quantify changes to neutrophil recruitment, ECM production and bone repair. Proteomic comparison of uninjured and fractured tissue from young and aged zebrafish, at time-points relevant to neutrophil recruitment, will highlight proteins warranting further study in neutrophil repair. Finally, as proof of concept for future studies, we will demonstrate the utility of zebrafish for screening the effect of drugs, commonly prescribed to older patients, on fracture repair, by investigating the effect of glucocorticoid treatment on the neutrophil response to fracture. Our project will characterise a previously underappreciated role for neutrophils in fracture repair and investigate the effect of ageing on this process. Proof of principle studies will demonstrate the utility of zebrafish for characterising age-related disease pathophysiology, and for testing compounds which influence fracture healing. We will use data generated in this seed study for as the basis for future long-term interdisciplinary projects of high clinical relevance and patient impact.

Lay abstract: Osteoporosis is a disease which causes bones to become fragile and fracture (called a fragility fracture). 3 million people are living with osteoporosis in the UK, resulting in half a million fractures annually. Fragility fractures cause long-term pain, mobility issues and social isolation, and cost the UK £4.4 billion every year; as the ageing population grows, the cost of osteoporosis will rise significantly. As well as being age-related, osteoporosis can be caused by steroid medicines used to treat inflammatory diseases like arthritis. These people often have weakened immune systems which are less able to respond to injuries. This means fractures in aged and immunosuppressed people don’t heal as well. Current therapies to prevent or treat fragility fractures are limited. Therefore, there is an urgent need for more research to identify new treatments for people living with osteoporosis. The ability of bone to repair after fracture depends on multiple factors. Neutrophils, a type of immune cell, are known to be important in the earliest stages, where they help to prevent infection and kickstart repair. Recent research suggests that neutrophils may also help to stabilise fractures by making an emergency scaffold to support damaged bone. We want to better understand the role of neutrophils in bone repair, and whether the neutrophil response to fracture is affected by ageing. Zebrafish are good models for studying fracture, as their bones are maintained by the same cells as humans. We can study bone repair over time in zebrafish, because their tails are transparent and contain bones which are easily fractured. The low cost compared to mice makes them a preferred model for studying the effect of aging on bone repair. Additionally, different combinations of drugs are easily given to zebrafish via their tank water. Our research shows that zebrafish get more fractures as they age. We have also developed a method for studying fractures over time. We will use this method in young and old zebrafish, to study how neutrophils behave after fracture. We will test whether reduced neutrophil function, often seen in older or immunocompromised people, affects bone repair. Finally, we will test whether drugs which affect neutrophils alter fracture repair in zebrafish. Our experiments will reveal more about the role of neutrophils in bone ageing and repair. We will determine whether neutrophils should be researched further as a target for treating fragility fractures in older patients.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM;MUSCULOSKELETAL