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.

A 6-week carbon tetrachloride (CCl4) exposure period will be used to produce liver fibrosis in various transgenic mouse lines. The ability of Nrf2 activation to inhibit fibrogenesis once initiated will be studied by treating the mice with TBE-31 (to pharmacologically activate Nrf2) or tamoxifen (to genetically activate Nrf2) over the last 3 weeks of CCl4 exposure. The ability of Nrf2 activation to accelerate resolution of liver fibrosis once injury has been curtailed will be studied by treating mice with TBE-31 or tamoxifen immediately after CCl4 exposure. In the first case, the effects of Nrf2 on fibrogenesis will focus on hepatic stellate cells (HSCs) by using the Pdgfrb gene promoter to allow both knockout and genetic activation of Nrf2. In the second case, the effects of Nrf2 on resolution of fibrosis will focus on liver monocyte-derived macrophages by using the Lys2 gene promoter. Upon sacrifice, HSCs or monocytes/macrophages will be prepared from the livers of these mice and determination of their trans-differentiation status and pro-inflammatory/resolution phenotypes measured by RNA-seq, using an NGS platform, to monitor expression of relevant genes. Thereafter, the HSCs and liver monocytes/macrophages will be cultured and the effects of Nrf2 on TGF-beta/PDGF signalling and the Nur-77 transcription factor in HSCs and monocytes/macrophages, respectively, examined by western blotting and by mass spectrometry (on an Agilent 1200 nanoLC-Orbitrap) to monitor oxidation of active site Cys residues in PTPs, PTEN and TXN1.

In translational experiments, we will examine whether in macrophages collected from patients with cirrhosis, in vitro activation of Nrf2 stimulates a pro-resolving phenotype using RNA-seq to follow gene expression and MSD V-plex immunoassay to measure cytokines production. Also, we will examine whether in HSCs collected from cirrhotic livers, in vitro activation of Nrf2 can cause reversal of the myofibroblast phenotype using qRT-PCR assays.

Patients with liver cirrhosis and the NHS, will both benefit from our proposed research. Chronic fibrotic liver disease is the 5th most common form of death in the UK and the incidence is rising; it is the second commonest cause of death in the under 65's. This is caused largely by alcohol consumption, viral hepatitis and obesity. It is estimated currently that 2% of the UK population have cirrhosis. This will rise due to Non-alcoholic Fatty liver disease (NAFLD). It is estimated that 25% of the adult UK population has fatty liver as a consequence of the marked increase in obesity and type II diabetes mellitus. Of these, at least 10% (i.e., 2.5% of the adult population) are thought to have NASH, a significant percentage of whom will progress on to develop liver cirrhosis and ultimately end-stage liver disease. At present, there is no effective treatment for cirrhosis other than liver transplantation. Thus, an effective therapy would have great clinical and commercial impact. The potential cost implications to the NHS of a rapidly rising incidence of cirrhosis is truly grave.

Strategies to treat liver cirrhosis (besides transplantation) are urgently required. Evidence suggests that transcription factor Nrf2 plays a pivotal role in inhibiting established and continuing fibrosis and/or increasing reversal of fibrosis. Importantly, Nrf2 is suppressed during development of cirrhosis. The experiments we describe will allow us to test in mice whether the in vivo activation of Nrf2 in hepatic stellate cells (HSCs) blocks ongoing fibrogenesis by suppressing TGFb and PDGF signaling as a consequence of antagonizing the redox signaling pathways that TGFb and PDGF rely on in order to cause trans-differentiation of HSCs into myofibroblasts that synthesize the fibrous scar material. Also, the experiments proposed will allow us to test in mice whether in vivo activation of Nrf2 in monocyte-derived macrophages accelerates the resolution of liver fibrosis by causing a switch in macrophage phenotype through antagonizing oxidative stress and redox signaling, which repress nuclear receptor Nur77 that contributes to regulation of macrophage differentiation. In translational studies, we will test whether in macrophages collected from patients with liver cirrhosis, the in vitro activation of Nrf2 causes a switch from a proinflammatory to a pro-resolving phenotype, and an increase in Nur77, and we will also test whether in HSCs collected from the livers of patients with cirrhosis, the in vitro activation of Nrf2 counters downregulation of Nrf2 that accompanies cirrhosis and so dampens the redox signaling that TGFb and PDGF require to stimulate fibrogenesis. Lastly, we will examine in archived fixed human liver from patients with cirrhosis whether downregulation of Nrf2 is associated with increased TGFb signaling and loss of nuclear Nur77, and if this downregulation in Nrf2 is associated with increases in levels of known negative regulators of Nrf2.

The commercial private sector, Biotechnology diagnostics and the Pharmaceutical industry, will all benefit from our research. Firstly, identification of molecular mechanisms, such as repression of TGFb/PDGF signaling and promotion of Nur77 activity, by which Nrf2 activation of Nrf2 ameliorates pathophysiological processes will allow other diseases besides liver fibrosis to be considered for Nrf2-targeted therapies. Secondly, a number of Pharmaceutical companies, have programmes aimed at developing Nrf2 activators, and they will be interested in the biochemical and clinical data produced by this project (see Cuadrado et al. Nat Rev Drug Disc 2019;18:295). Thirdly, staff employed on this project grant will gain skills in general molecular cell biology techniques, mouse genetics and disease models, gene expression profiling, redox proteomics and redox signaling, all of which are likely to be highly valued by the pharmaceutical industry and would therefore increase their employment prospects.