Role of LAP in controlling liver homeostasis

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.

Clearance of extracellular debris and dying cells is essential to preserve multicellular tissue homeostasis, particularly in the liver where low-grade apoptosis and replacement of dead hepatocytes occurs by regeneration.

Very recently, a new pathway was described that mediates degradation of extracellular material by phagocytic cells, the LC3 associated phagocytosis (LAP) pathway that uses components of the autophagy pathway to conjugate autophagy protein LC3 to phagosomes, thus facilitating fusion with lysosomes and cargo degradation.

Our team led research demonstrating that the dWD domain of ATG16L1 is essential for activation of LAP. We also showed, in genetically-modified mice (which we generated) both with LAP depleted systemically or specifically in myeloid cells, that LAP is key to preservation of brain homeostasis and protection of the lung from influenza. Despite this, the role of LAP in hepatocytes remains unknown.

This project will define the role of LAP in hepatocytes for regulation of liver responses to injury and regeneration.
We will characterise the LAP machinery in primary hepatocytes, define the stimuli triggering LAP, and determine the functional consequences of its activation in these cells. Next, we will define how LAP in hepatocytes influences the liver response to acute and chronic hepatocellular injury in wild-type and LAP-hepatocyte-deficient mice and how it influences the function of all the different liver cells using high-throughput single cell sequencing technology. Ultimately, we will determine how LAP modulates liver regeneration after challenge via modulation of hepatocyte proliferation.

We anticipate providing new fundamental knowledge on how LAP in hepatocytes modulates liver responses to injury. This will inform future research to improve liver responses to injury and prevent/treat disease progression by targeting LAP in a cell-specific manner.