Role of LAP in controlling liver homeostasis

死细胞清除是一个基本的生理过程，对整个生命的组织稳态和对多细胞组织损伤的有效反应至关重要；通常被称为吞噬作用，它是由专业（免疫、骨髓）和非专业（上皮、内皮）吞噬细胞实现的，这些吞噬细胞识别、内化和降解垂死细胞和细胞外碎片。最近，新的lc3依赖性吞噬（LAP）途径被描述为介导细胞外成分的吞噬及其细胞内加工和降解。我们的研究小组（合作i: Wileman）已经定义了调节LAP的分子成分，以及它如何保持大脑稳态并预防老年阿尔茨海默病。我们的团队还在其他组织中剖析了细胞特异性LAP功能，显示了LAP激活如何影响肺部对流感病毒感染的敏感性。为了提高我们对肝脏细胞清除的理解，我们培育了一种转基因小鼠，在这种小鼠中，LAP可以在肝细胞中被特异性地耗尽。这种独特的实验工具使我们能够解剖LAP在肝细胞中的未知作用及其对肝功能的影响。这一点尤其重要，因为肝脏不断受到来自肠道循环的抗原和细菌产物的挑战，以及来自血液循环的垂死淋巴细胞的涌入。这些细胞促进肝细胞的低级别凋亡，并在增殖后被新的肝细胞所取代，这是一个由巨噬细胞相互作用密切调节的再生过程。在肝损伤后肝细胞过度死亡的反应中，这种自我调节的相互作用可能变得不堪重负，导致慢性炎症和疾病进展。值得注意的是，尽管肝细胞吞噬死细胞的能力众所周知，但其基本机制仍不明确，这支持了我们迫切需要进行旨在表征肝细胞LAP并确定其对肝功能和损伤反应的影响的研究。我们使用肝细胞特异性LAP缺陷小鼠的试验结果表明，LAP缺失可保护肝脏免受肝细胞死亡、炎症和纤维化。这表明LAP在肝细胞中具有特异性功能，不同于最近在浸润肝脏的髓细胞中描述的功能，并支持LAP在肝细胞调节肝脏对挑战的反应中的关键作用。在我们激动人心的试点工作的基础上，我们提出了一项研究计划，将小鼠肝细胞和组织样本的体外和体内工作结合起来，以确定LAP在肝细胞中调节肝功能和免疫的具体作用。首先，我们将定义是什么激活了肝细胞中的LAP；表征LAP途径的分子机制；并利用我们实验室成熟的体外技术确定肝细胞内货物的命运。接下来，我们将利用我们独特的转基因小鼠和实验模型，确定肝细胞lap如何控制肝脏对损伤和炎症的体内反应，这些模型也在我们的小组中得到了证实。利用我们团队的专业知识，我们将使用高通量10倍单细胞测序技术精确分析肝细胞lap激活如何影响其他个体肝细胞类型在损伤反应期间的功能。最后，我们将运用我们在肝脏再生机制方面的专业知识来确定LAP在挑战后肝脏功能恢复和肝脏稳态保存中的作用。通过这一研究项目，我们将对肝细胞lap在控制肝脏损伤反应和预防疾病进展中的作用提供新的基本机制见解。这将使我们能够制定未来的战略，减少肝细胞损伤，从而预防和治疗疾病，保持终身健康。

Dead cell clearance is a fundamental physiological process essential for tissue homeostasis throughout life, and for efficient responses to multicellular tissue damage; generally termed phagocytosis, it is achieved by professional (immune, myeloid) and non-professional (epithelial, endothelial) phagocytic cells that recognise, internalise and degrade dying cells and extracellular debris.Recently, the novel LC3-dependent phagocytosis (LAP) pathway was described which mediates engulfment of extracellular components and their intracellular processing and degradation. Our research team (co-I: Wileman) has defined the molecular components regulating LAP and how this preserves brain homeostasis and prevents Alzheimer's Disease during old age. Our team also dissected cell-specific LAP functions in other tissues, showing how LAP activation affects sensitivity to influenza virus infection in the lung.To advance our understanding of cell clearance in the liver, we have generated a transgenic mouse in which LAP can be depleted specifically in hepatocytes. This unique experimental tool enables us to dissect the, as yet unknown, role of LAP in hepatocytes and its influence on liver function.This is particularly important since the liver is constantly challenged with antigens and bacterial products circulating from the intestine, as well as an influx of dying lymphocytes from blood circulation. These promote low-grade apoptosis of hepatocytes which are replaced by new hepatocytes after proliferation, a regenerative process tightly regulated by crosstalk with resident macrophages. In response to excessive hepatocyte death after liver injury, this self-regulated interaction can become overwhelmed leading to chronic inflammation and disease progression.Remarkably, despite the well-known capacity of hepatocytes to engulf dead cells, the underpinning mechanisms remain undefined, supporting the urgent need for our research aimed at characterising LAP in hepatocytes and defining its influence on liver function and response to injury.Our pilot results using hepatocyte-specific LAP-deficient mice show that absence of LAP protects the liver from hepatocellular death, inflammation and fibrosis. This indicates that LAP has a specific function in hepatocytes, different from that recently described in myeloid cells infiltrating the liver, and supporting the key role of LAP in hepatocyte modulation of liver response to challenge.Building on our exciting pilot work, here we propose a programme of research that integrates in vitro and in vivo work in murine liver cells and tissue samples, to define the specific role of LAP in hepatocytes for regulation of liver function and immunity.First, we will: define what activates LAP in hepatocytes; characterise the molecular machinery of the LAP pathway; and determine the fate of intracellular cargo in hepatocytes using in vitro techniques that are well-established in our labs.Next, we will determine how hepatocyte-LAP controls liver responses to injury and inflammation in vivo using our unique genetically-modified mice and experimental models, also well-established in our groups. Exploiting the expertise of our team, we will precisely dissect how hepatocyte-LAP activation can influence on other individual liver cell type function during responses to injury using high-throughput 10x single cell sequencing technology.Ultimately, we will apply our expertise in the mechanisms mediating liver regeneration to define the role of LAP in restoration of liver function after challenge and preservation of liver homeostasis.Through this programme of research, we will deliver new fundamental mechanistic insights into the role of hepatocyte-LAP in controlling liver responses to injury and in preventing disease progression. This will enable us to develop future strategies that will reduce hepatocyte injury and thus prevent and treat disease and preserve life-long health.