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

在人类和动物中，高活性的中性粒细胞反应与传染病、炎症性疾病和创伤有关。中性粒细胞对于保护宿主免受入侵病原体的侵袭和协调炎症至关重要。然而，如果在错误的时间、错误的地点或错误的程度激活，它们可能会损害宿主和/或引发严重的炎症。对于以中性粒细胞驱动的炎症为特征的疾病，显然需要有效的策略来重新编程过度活跃的反应。抑制性受体是人和动物中性粒细胞表达的关键分子，可以抑制它们的激活，确保它们不会过早或过度激活。缺乏抑制性受体的小鼠对炎症和传染病的易感性增加，证明了它们在维持健康方面的重要性。靶向抑制性受体是抑制高活性中性粒细胞反应的一种有前景的策略。然而，协调中性粒细胞调节的单个抑制性受体还不完全清楚。这一点意义重大，因为我们可能没有意识到抑制性受体为开发新的免疫抑制疗法提供了巨大的潜力。LILRB3是一种表达在人中性粒细胞上的抑制性受体，是一种有效的激活、脱颗粒、吞噬和微生物杀伤的抑制剂。重要的是，LILRB3在循环中的中性粒细胞上高度表达。这与大多数抑制性受体的低表达水平形成对比，中性粒细胞激活时受体表达上调。因此，LILRB3是一组独特的抑制性受体，可以在疾病情况下靶向关闭中性粒细胞的动员、激活和高反应。值得注意的是，LILRB3的体内功能、信号机制和结构尚不清楚。因此，在LILRB3维持人类健康的过程中，我们对中性粒细胞是如何调节的知识存在着一个根本的空白。我们需要了解LILRB3的生理作用，以了解它是如何协调复杂系统中的中性粒细胞来维持健康的。我们需要定义LILRB3信号来认识它是如何抑制中性粒细胞反应的。最后，我们需要描述LILRB3的结构，以便了解它是如何发挥作用的，以及它如何成为治疗的靶点。我已经确定了第一个LILRB3配体的特征，它是一种称为B的细菌表面蛋白。B中的两个结构域与LILRB3高亲和力结合并具有激动性。因此，B为研究LILRB3的生物学特性提供了一个有用的实验系统。这个项目将提供对LILRB3在中性粒细胞上的生理作用的见解，信号通路和结构，使用我的团队确定的新的细菌来源的LILRB3配体。具体地说，在这个为期三年的项目中，我们将A)利用新开发的转基因人类LILRB3小鼠确定LILRB3在中性粒细胞成熟、炎症和宿主防御中的作用，B)表征中性粒细胞中的LILRB3信号，以及C)解析LILRB3的结构，并识别新的病原体来源的LILRB3配体。数据将提供LILRB3在调节中性粒细胞成熟、炎症和宿主防御中的作用的详细信息。重要的是，该项目将提供有关LILRB3的第一个结构信息和关于中性粒细胞中LILRB3信号的第一个详细信息。这些实验资源、技术和数据将适用于其他白细胞，如巨噬细胞和其他动物系统。长期目标是在分子和系统水平上了解LILRB3如何在宿主防御过程中协调中性粒细胞反应，扩大我们对维持终身健康的基本免疫过程的理解。这将为以LILRB3为靶点的免疫疗法的发展铺平道路，以抑制疾病情况下的中性粒细胞反应。

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.