Immune (dys-)regulation by the PD-1/PD-L checkpoint pathway in chronic liver disease

慢性肝病(CLD)是一种全球性的医疗负担，全世界有超过8.4亿人受到影响，而且发病率还在上升。不管慢性肝病的病因是什么，持续的肝脏损伤(例如酒精)会导致瘢痕组织的逐渐形成(称为纤维化)、肝功能丧失，并可能最终导致肝硬化(疤痕形成)。肝硬变患者的主要临床并发症和/或感染的急性发展称为急性肝硬变失代偿。非酒精性脂肪性肝病(NAFLD)是CLD的另一种常见病因，在全球25%的成年人中存在。NAFLD与肥胖和2型糖尿病有关，当肝脏中沉积了太多脂肪时就会发生。这与纤维化、进展为肝硬变和增加患肝癌的风险有关。治疗选择有限，因此开发新的有效治疗方法具有巨大的临床需求。免疫系统在CLD的进展中起着至关重要的作用。巨噬细胞是存在于我们全身的免疫细胞。肝巨噬细胞在其表面表达不同的蛋白质，通过这些蛋白质与其他细胞相互作用(“串扰”)，或感受来自组织损伤和脂肪或疤痕组织沉积的信号。因此，巨噬细胞在调节肝脏损伤中起着中心作用。随着疾病进展到肝硬变，这些信号可能会降低免疫细胞的功能，从而增加患者对微生物感染的易感性和死亡风险。我们最近的工作研究了一条关键的细胞信号通路，免疫细胞通过其表面的PD-1和PD-L蛋白进行沟通。我们发现，该途径控制着肝巨噬细胞的功能，其治疗靶向可以提高急性肝损伤后巨噬细胞的抗微生物反应。我们现在打算更详细地研究这一途径，以了解它如何在慢性肝损伤后控制免疫细胞的功能。为了实现这一目标，我们将使用现代技术方法在人类和老鼠样本中进行一系列实验。首先，我们将使用流式细胞术和多光谱成像的科学技术，并将其应用于CLD患者的血液和肝组织样本。这将使我们能够检测哪些免疫细胞表达PD-1和PD-L表面蛋白。接下来，我们将使用细胞培养和流式细胞术来研究这些蛋白质的相互作用如何改变免疫细胞功能。我们将使用患者的血浆和粪便样本进行细胞培养实验，以确定增加PD-1/PD-L蛋白水平的信号。然后，我们将使用抑制这些信号的治疗药物，旨在恢复CLD患者的免疫细胞功能(例如，增强抗菌防御)。其次，我们的目标是确定在慢性肝损伤过程中，随着时间的推移，哪些肝细胞表达PD-1和PD-L蛋白。为此，我们将使用小鼠的肝纤维化和脂肪肝模型，并对从小鼠的血液和肝脏中分离的细胞进行流式细胞术。接下来，我们将评估当PD-L蛋白没有产生时，肝脏损伤是改善还是恶化。为了实现这一点，我们将使用肝巨噬细胞不表达PD-L蛋白的转基因小鼠。我们将对肝细胞样本进行一种名为单细胞RNA测序的新技术，这将告诉我们损伤后免疫细胞的功能是如何变化的。最后，在确定了哪些信号会增加PD-1/PD-L蛋白水平后，我们将使用药物来阻断这些信号，并研究它们的治疗靶向是否可以减少肝纤维化和/或提高肝巨噬细胞介导的抗菌防御。总的来说，通过完成这个项目，我们将增加我们对PD-1/PD-L通路如何调控免疫细胞功能从而调节肝损伤的理解。这将为治疗干预提供新的机会，发现CLD进展的预后生物标志物，并最终改善患者的预后。

Chronic liver disease (CLD) is a global healthcare burden, affecting over 840 million people worldwide and has a rising incidence. Irrespective of the cause of CLD, persistent damage to the liver (e.g., alcohol) leads to the progressive formation of scar tissue (called fibrosis), loss of liver function, and may ultimately result in cirrhosis (scarring). The acute development of major clinical complications and/or infections in cirrhotic patients is called acute decompensation of cirrhosis. Non-alcoholic fatty liver disease (NAFLD), another common aetiology of CLD, is present in 25% of adult population globally. NAFLD is associated with obesity and type 2 diabetes and arises when too much fat is deposited in the liver. This is linked to fibrosis, progression to cirrhosis and increased risk of liver cancer. Treatment options are limited so there is a huge clinical need to develop new effective therapies.The immune system plays a crucial role in CLD progression. Macrophages are immune cells present throughout our body. Liver macrophages express different proteins on their surface via which they interact ("cross-talk") with other cells or sense signals from tissue damage and the deposition of fat or scar tissue. Hence, macrophages play a central part in regulating liver injury. As disease advances to cirrhosis, these signals may reduce the function of immune cells, thus increasing patients' susceptibility to microbial infections and mortality risk.Our recent work studied a crucial cell signalling pathway via which immune cells use the PD-1 and PD-L proteins on their surface to communicate. We showed that this pathway controls the function of liver macrophages, and its therapeutic targeting can improve macrophage anti-microbial responses after acute liver injury. We now intend to study this pathway in more detail, to understand how it controls the function of immune cells after chronic liver injury. To achieve this, we will perform series of experiments using modern technological approaches in human and mouse samples. Firstly, we will use scientific techniques called flow cytometry and multispectral imaging and apply them on blood and liver tissue samples of CLD patients. This will allow us to detect which immune cells express the PD-1 and PD-L surface proteins. We will next use cell culture and flow cytometry to study how these proteins' interactions alter immune cell function. We will use patient plasma and stool samples to perform cell culture experiments to identify the signals that increase PD-1/PD-L protein levels. We will then use therapeutic drugs that inhibit these signals, aiming to restore the function of immune cells from CLD patients (e.g., to enhance antimicrobial defence). Secondly, we aim to identify which liver cells express PD-1 and PD-L proteins over time during chronic liver injury. To do this, we will use mouse models of liver fibrosis and fatty liver disease and perform flow cytometry on cells isolated from the blood and livers of mice. We will next assess if liver injury improves or gets worse when the PD-L protein isn't produced. To achieve this, we will use genetically-modified mice whose liver macrophages do not express the PD-L protein. We will perform a new technique called single-cell RNA sequencing on liver cell samples, and this will tell us how immune cells' function changes after injury. Finally, having identified which signals increase PD-1/PD-L protein levels, we will use drugs to block these signals and study if their therapeutic targeting can reduce liver fibrosis and/or improve the antimicrobial defence mediated by liver macrophages.Overall, by completing this project we will increase our understanding of how the PD-1/PD-L pathway controls immune cell function and therefore regulates liver injury. This will provide new opportunities for therapeutic intervention, discovery of prognostic biomarkers for CLD progression, and ultimately enhance patient outcomes.