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

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.

Chronic liver disease (CLD) affects over 840 million people globally. Decompensated cirrhosis and non-alcoholic fatty liver disease are common CLDs linked with fibrosis. Treatment options are limited so there is a huge clinical need to develop new therapies. Liver macrophages integrate signals from the liver, gut or overnutrition, thus play central parts in regulation of inflammation and fibrosis progression. CLDs are associated with impaired antimicrobial responses which contribute to high risk of infections and mortality.

Our recent work revealed a key role for the PD-(L)1 pathway in regulating macrophages after acute liver injury. Here, we will study how this axis contributes to immune-mediated control of CLD. We will use human/translational and mouse models of chronic liver injury with cutting-edge technological approaches to study the immune-regulatory role of the PD-1/PD-L axis in CLD conditions and investigate its therapeutic modulation.

The proposed experiments will assess PD-1/PD-L expression on immune cells in blood and liver CLD patient samples by multispectral flow cytometry and tissue staining. We will use cellular and molecular techniques and patient samples (e.g., plasma) in vitro to identify inflammatory cues inducing axis' upregulation. We will study its signalling activation status, mechanism of action, and its targeting (e.g., TLR or cytokine inhibition). We will further use novel transgenic animal models and state-of-the-art platforms (scRNA-seq and intravital imaging) to determine in vivo the axis' immune-regulatory role after injury, how it's influenced by gut microbiome, and assess its targeting for improving antimicrobial responses in CLD.

This work will improve our knowledge of the PD-1/PD-L control of immune-regulated liver fibrosis and antimicrobial responses. It will thus provide new opportunities for therapeutic strategies, discovery of novel prognostic biomarkers for CLD progression, and may ultimately improve patient outcomes