Characterising the Physiological Role, Function and Structure of the Inhibitory LILRB3 Receptor on Neutrophils using a Novel Bacterial Ligand

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Hyper-active neutrophil responses are associated with infectious disease, inflammatory disease and trauma in humans and animals. Neutrophils are critical for protecting hosts from invading pathogens and coordinating inflammation. However, if activated at the wrong time, wrong place or to the wrong extent they can damage the host and/or induce severe inflammation. Effective strategies to reprogram hyperactive responses are clearly needed for diseases characterised by neutrophil-driven inflammation. Inhibitory receptors are key molecules expressed by human and animal neutrophils that suppress their activation and ensure that they are not activated prematurely or excessively. Their importance in maintaining health is demonstrated by the increased susceptibility of mice that lack inhibitory receptors to inflammatory and infectious diseases. Targeting inhibitory receptors is a promising strategy to suppress hyper-active neutrophil responses. However, the individual inhibitory receptors that coordinate neutrophil regulation are not fully understood. This is significant as we are likely unaware of the considerable potential that inhibitory receptors offer for the development of novel immunosuppressive therapies.LILRB3 is an inhibitory receptor expressed on human neutrophils that is a potent inhibitor of activation, degranulation, phagocytosis and microbial killing. Importantly, LILRB3 is highly expressed on circulating neutrophils. This contrasts the low expression levels of most inhibitory receptors, that are upregulated upon neutrophil activation. Therefore, LILRB3 is in a unique group of inhibitory receptors that could be targeted to switch off neutrophil mobilisation, activation and hyper-responses in disease situations. Remarkably, the in vivo functions, signalling mechanisms and structure of LILRB3 are unknown. Thus, there is a fundamental gap in our knowledge of how neutrophils are regulated during maintenance of human health by LILRB3. We need to understand the physiological role of LILRB3 to appreciate how it coordinates neutrophils in complex systems to maintain health. We need to define LILRB3 signalling to recognise how it inhibits neutrophil responses. Finally, we need to characterise the structure of LILRB3 in order to understand how it functions and how it could be targeted therapeutically. I have characterised the first LILRB3 ligand, which is a bacterial surface protein called B. Two domains within B bind LILRB3 with high-affinity and have agonistic properties. Therefore, B provides a useful experimental system to probe the biology of LILRB3. This project will provide insights into the LILRB3 physiological role on neutrophils, signalling pathways and structure, using the novel bacteria derived LILRB3 ligands identified by my group. Specifically, during this three-year project we will A) define the role of LILRB3 in neutrophil maturation, inflammation and host defence using newly developed transgenic human LILRB3 mice, B) characterise LILRB3 signalling in neutrophils, and C) resolve the structure of LILRB3, and identify new pathogen derived LILRB3 ligands.Data will provide detailed information on the role of LILRB3 in regulating neutrophil maturation, inflammation and host defence. Importantly, the project will provide the first structural information on LILRB3 and the first detailed information on LILRB3 signalling in neutrophils. The experimental resources, techniques and data will have applicability to other leukocytes, such as macrophage, and other animal systems. The long-term goal is to understand at the molecular and system level how LILRB3 coordinates neutrophilic responses during host defence, expanding our understanding of fundamental immunological processes that maintain lifelong health. It will pave the way for development of LILRB3-targetting immunotherapeutics to suppress neutrophil responses in disease situations.