Therapeutic Targeting of Pathogenic Scar-associated Macrophages in the Fibrotic Niche of Chronic Liver Disease

肝病是一个主要的全球医疗保健问题，全球有8.44亿人受到影响，每年导致200万人死亡。令人担忧的病例数继续上升，预计肝病很快将成为英国过早死亡的最常见原因。有许多不同的肝脏疾病的原因。无论潜在的原因是什么，对肝脏的长期损害都会导致肝脏瘢痕形成，称为纤维化。随着时间的推移，纤维化的程度逐渐恶化，这意味着肝脏最终停止正常运作并开始衰竭。由于进行性瘢痕形成在促进肝衰竭和临床并发症的发展中的重要作用，人们对试图找到新的治疗方法来阻断这种瘢痕形成过程很感兴趣。这些抗纤维化疗法将是治疗患者的一个非常重要的步骤，但不幸的是，目前还没有这样的疗法。巨噬细胞是存在于全身的免疫细胞。我们以前已经表明，肝脏中的巨噬细胞在调节瘢痕形成过程中起着至关重要的作用。因此，试图阻断这些肝脏巨噬细胞的功能是一种有吸引力的尝试和减少纤维化的策略。我们最近的工作使用一种称为单细胞RNA测序的新技术研究了患病人类肝脏中的巨噬细胞。我们能够在疤痕肝脏中识别出一种独特类型的巨噬细胞，这对促进纤维化很重要。我们现在打算更详细地研究这些巨噬细胞，目的是找到抑制这些特定细胞功能的方法，并最终开发新的抗纤维化疗法。为了实现这一目标，我们将使用现代科学技术进行一系列实验。首先，为了获得关于促进纤维化的巨噬细胞在肝脏疾病进展过程中如何发挥作用的更准确信息，我们将邀请正在接受肝脏活检的患者作为计划的医院调查的一部分，将该活检的一小部分捐赠给我们的研究。我们将使用这种组织进行单细胞RNA测序，以了解巨噬细胞在纤维化发展过程中的行为。我们还将在这些活检中使用另一种称为空间转录组学的新科学技术，告诉我们这些巨噬细胞如何与肝脏中的相邻细胞交谈以调节瘢痕形成过程，并帮助我们确定这些功能可能被抑制的潜在方式。其次，我们打算更好地了解这些巨噬细胞如何随着时间的推移而变化，比较它们在暴露于进一步肝损伤或损伤停止时的行为。为了做到这一点，我们将使用肝脏瘢痕形成的小鼠模型，其中巨噬细胞与患者中发现的巨噬细胞非常相似，但我们能够在日常基础上研究这些细胞。这些信息将使我们能够确定促进进行性瘢痕形成的巨噬细胞与肝脏中的其他巨噬细胞有何不同，从而确定我们如何特异性靶向它们。最后，在确定了这些巨噬细胞产生的特定基因和蛋白质后，我们将利用这些新信息来尝试阻断这些细胞的功能并抑制肝脏中的瘢痕形成。最初，这将使用一种名为CRISPR的技术来完成，这将使我们能够快速同时评估肝脏巨噬细胞中众多基因的功能。然后，我们将专注于我们所证明的重要的特定基因，使用小鼠模型和人类细胞来测试阻断这些分子是否有助于减少纤维化。最终，在完成这个项目时，我们希望确定新的策略来抑制肝脏中促进纤维化的巨噬细胞的功能，然后可以开发为肝脏疾病患者急需的抗纤维化疗法。

Liver disease is a major global healthcare problem, with 844 million people thought to be affected worldwide resulting in 2 million deaths each year. Worryingly case numbers continue to rise, and liver disease is soon predicted to become the commonest cause of premature death in the UK. There are many different causes of liver disease. Irrespective of the underlying cause, long-term damage to the liver leads to the development of liver scarring, called fibrosis. The amount of fibrosis gets progressively worse over time, meaning that the liver eventually stops functioning properly and starts to fail. Due to the important role of progressive scarring in promoting liver failure and the development of clinical complications, there is a lot of interest in trying to find new treatments to block this scarring process. These antifibrotic therapies would be a very important step forward in the treatment of patients, but unfortunately no such therapies are currently available.Macrophages are immune cells present throughout the body. We have previously shown that macrophages in the liver play a crucial role in regulating the scarring process. Trying to block the function of these liver macrophages is therefore an attractive strategy to try and reduce fibrosis. Our recent work has studied macrophages in the diseased human liver using a new technology called single-cell RNA-sequencing. We were able to identify a unique type of macrophage in the scarred liver which is important for promoting fibrosis. We now intend to study these macrophages in more detail, with the aim of finding ways of inhibiting the function of these particular cells and ultimately developing new antifibrotic therapies.In order to achieve this, we will perform a series of experiments using modern scientific techniques. Firstly, in order to get more precise information on how the fibrosis-promoting macrophages function during the progression of liver disease, we will invite patients who are undergoing a biopsy of their liver as part of their planned hospital investigations, to donate a small excess portion of this biopsy to our research study. We will use this tissue to perform single-cell RNA-sequencing to understand how macrophages behave during the development of fibrosis. We will also use another new scientific technique called spatial transcriptomics on these biopsies, to tell us how these macrophages might be talking to neighbouring cells in the liver to regulate the scarring process and help us to identify potential ways these functions can be inhibited. Secondly, we intend to better understand how these macrophages change over time, comparing how they behave when they are exposed to further liver damage or when the damage is stopped. In order to do this, we will use mouse models of liver scarring where the macrophages are very similar to those found in patients, but we are able to study these cells on a more day-to-day basis. This information will enable us to determine how the macrophages which promote progressive scarring differ from other macrophages in the liver and therefore how we might specifically target them. Finally, having identified the specific genes and proteins made by these macrophages, we will use this new information to try and block the function of these cells and inhibit scarring in the liver. Initially this will be done using a technology called CRISPR, which will enable us to rapidly and simultaneously assess the functions of numerous genes in liver macrophages. We will then focus on the specific genes which we show to be important, using both mouse models and human cells to test whether blocking these molecules will be useful to reduce fibrosis.Ultimately, in completing this project we expect to identify new strategies to inhibit the function of fibrosis-promoting macrophages in the liver, which can then be developed as badly-needed antifibrotic therapies for patients with liver disease.