Defining the oxidative stress-related mechanisms by which activation of the transcription factor Nrf2 arrests and resolves liver fibrosis

Repetitive wound healing in the liver results in fibrosis leading to scarring and, if severe, to cirrhosis. It is a serious condition that can lead to liver failure, and liver cancer. Liver cirrhosis arises due to a variety of causes including viruses, alcohol, auto-immunity, metabolic conditions and obesity-related liver damage. There has been a huge increase in cirrhosis, which poses an enormous challenge to our NHS. Most alarmingly, there is currently no effective therapy for liver fibrosis, except transplantation. Recently we discovered that by activating a major regulator of intracellular antioxidant systems, called Nrf2, we were able to arrest and/or reverse established and ongoing liver fibrosis in the mouse. The purpose of this project is to determine how activation of Nrf2 changes the formation and removal of liver scar tissue. The scarring is laid down by specific cells in the liver called hepatic stellate cells (HSCs), and is removed by immune cells recruited into the liver called macrophages. An important feature of liver fibrosis is that its initiation, perpetuation and progression involve cooperation between different cell types within the liver, including the immune cells, which is governed by production of various signalling molecules that allow communication between the different cell types. One such class of molecule are oxidants, called reactive oxygen species (ROS), that trigger pro-fibrotic and pro-inflammatory signalling cascades. As the development of liver fibrosis involves production by HSCs of ROS, which trigger redox signalling that causes their differentiation into collagen-secreting myofibroblasts that proliferate, we envisage that activation of Nrf2 in HSCs blocks synthesis of fibrous scar tissue in the liver by inducing antioxidant genes that inactivate ROS and block redox signalling. ROS also simulate inflammatory responses in macrophages recruited into the liver, so rather than allowing them to kill HSCs and digest extracellular scar material, they cause more damage, we envisage that activation of Nrf2 in these immune cells dampens inflammation and promotes resolution of fibrosis by inducing antioxidant genes that inactivate ROS and block redox signalling, allowing the cell to resolve the fibrosis. To evaluate these hypotheses, we will use transgenic mice to explore whether pharmacological and genetic activation of Nrf2 in HSCs of mice with ongoing liver fibrosis arrests synthesis of fibrous scar protein in the liver by blunting redox signalling and will identify which of the genes regulated by Nrf2 contributes to inhibition of fibrogenesis. In a similar manner, we will use transgenic mice to explore whether pharmacological and genetic activation of Nrf2 in immune cells of mice that have liver fibrosis but in which liver injury has ceased, accelerates removal of the fibrous scar by blunting redox signalling, and will identify which of the genes regulated by Nrf2 contribute to resolution of fibrosis. To examine the clinical significance of results we will examine the abundance of Nrf2 in HSCs isolated from the livers of patients with cirrhosis and evaluate whether activation of Nrf2 induces target genes, suppresses ROS levels and inhibits the synthesis of fibrous protein. Also, we will take macrophages from the blood of patients with cirrhosis and see if activation of Nrf2 induces Nrf2-target genes, suppresses ROS levels and promotes an anti-inflammatory and pro-resolving cell type. Lastly, we will evaluate the value of Nrf2 measurements in the liver of patients with liver disease to predict clinical outcome and shed light on the molecular processes that lead to liver fibrosis. Thus, we will measure Nrf2 levels in archived liver sections from patients with liver disease and assess whether Nrf2 is downregulated and expression of its target genes suppressed in severe cases of liver cirrhosis, and explore whether proteins that are known to be negative regulators of Nrf2 are upregulated.