Function of IKKbeta in the progression and regression of liver fibrosis

Lead Research Organisation: Newcastle University
Department Name: Institute of Cellular Medicine

Abstract

Liver cirrhosis is the end stage of a process known as fibrosis in which the normal healthy tissues of the liver are over several years progressively replaced with non-functional scar tissue. This process leaves the liver in a state of structural and functional disorder for which the only option is replacement by transplantation to prevent death. This disease process can be caused by a wide variety of injuries including those inflicted by viruses (hepatitis C and B), alcohol, metabolic agents and autoimmune attack. Recent work in the applicants laboratory as well as in other laboratories across the world has given new hope to the treatment of liver fibrosis by demonstrating that it is a process that can be reversed. Reversibility of fibrosis is associated with the removal from the liver of a specialised cell (hepatic myofibroblast) that is responsible for production of scar tissue, however removal of these cells is very inefficient in livers that are undergoing persistent injury. In pioneering studies the applicants have shown that drugs which target a survival factor called NF-kB will promote the removal of hepatic myofibroblasts from the liver without affecting other liver cells required for healthy function. The aim of the project for which grant income is requested is to learn more about one of the key molecules that control NF-kB. This molecule known as IKKbeta is already a target for emerging anti-inflammatory drugs which may eventually offer an alternative to transplantation if we can find evidence that it plays an important role in fibrosis. The experiments that are proposed are designed to discover if loss of IKKbeta and NF-kB activities in hepatic myofibroblasts will result in reduced severity of fibrosis and enhanced rates of recovery from liver disease. The results will be used to inform colleagues in hepatology clinics as to the likely benefits of the use of IKKbeta inhibitors in the treatment of liver disease patients. The results of the work will also be presented to the general public via press releases (organised through the University of Southampton and the Medical Research Council Press Offices and Websites) as well as in the form of public lectures and publication of data in high quality scientific journals.

Technical Summary

Liver fibrosis involves progressive replacement of liver tissue with scar tissue in response to chronic injury (alcohol, HCV, fat etc) and leads to the end-stage disease of cirrhosis. In the past 5 years, clinical and experimental studies have revealed a new paradigm, that fibrosis can regress as well as progress. However, spontaneous regression is only achieved in a small number of patients that can be effectively treated for the underlying cause of liver disease and even in these cases recovery is slow and incomplete. Studies pioneered by the applicants shows that stimulation of apoptosis of scar producing hepatic myofibroblasts (HM) accelerates the rate at which experimental fibrosis is spontaneously resolved. These studies identified NF-kB and its regulatory IkappaKinases (IKK) complex as critical controllers of HM apoptosis. Inhibition of NF-kB/IKK activity is associated with elevated rates of HM apoptosis and rapid recovery from fibrosis. The overarching aim of the current proposal is to determine the role played by the IKKbeta component of the IKK complex and to develop a highly specific inhibitor of IKKbeta action in HM. The role of IKKbeta in both the progressive and regressive phases of fibrosis will be investigated using a mouse in which the IKKbeta gene can be deleted at will and selectively in fibroblasts and myofibroblasts. Mice in which the IKKbeta allele is flanked by loxP sites will be crossed with mice in which Cre is under the transcriptional control of the alpha2 (I) collagen enhancer and post-translational control by Tamoxifen providing regulated and cell-specific knockout of IKKbeta expression. Importantly, this system will enable the fibrogenic role of IKKbeta to be studied independently from its pro-inflammatory function in the injured liver. The mice will be used in models of fibrogenesis (CCl4 injury) and recovery from fibrosis (following chronic injury with CCl4). Histological (Sirius Red for collagen I/III and alphaSMA for numbers of HM) and biochemical (qRT-PCR for fibrogenic markers, hydroxyproline and MMP activity assays). We anticipate that loss of IKKbeta will be associated with prevention of fibrosis and improved rates of recovery from fibrosis. Work in the second part of the proposal will address the way in which IKKbeta regulates NF-kB which from previous work and pilot data appears to operate via phosphorylation of the p65 subunit of NF-kB. In addition a specific peptide inhibitor of IKKbeta-mediated p65 phosphorylation will be tested for its ability to inhibit NF-kB, promote apoptosis and stimulate recovery from fibrosis.

Publications

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Campbell SJ (2008) Hepatic nuclear factor kappa B regulates neutrophil recruitment to the injured brain. in Journal of neuropathology and experimental neurology

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Gaspar-Pereira S (2012) The NF-?B subunit c-Rel stimulates cardiac hypertrophy and fibrosis. in The American journal of pathology

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Hagens WI (2008) Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers. in The Journal of pharmacology and experimental therapeutics

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Hanley KP (2008) Ectopic SOX9 mediates extracellular matrix deposition characteristic of organ fibrosis. in The Journal of biological chemistry

 
Description MRC Programme
Amount £1,904,040 (GBP)
Funding ID MR/K001949/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 03/2017
 
Description NIAAA, NIH
Amount £400,000 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 12/2011 
End 01/2016
 
Description NIHR/MRC EME
Amount £1,400,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2011 
End 01/2013
 
Title Col1-IKKbeta targeted knockout mouse 
Description A new transgenic mouse was developed that generates conditional knockout of the IKKbeta gene in cells expressing collagen I. The mouse is currently under examination. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact None as yet 
 
Title nfkb1S240A mouse 
Description Work on IKK and NF-kB in the lab discovered a Serine residue in the nfkb1 gene that controls how its protein product p50 selects dimer partners. The nfkb1S340A mouse carries a mutation at this residue. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The mouse helped us determine that p50:p50 NF-kB homidimers function as suppressors of liver cancer by halting the recruitment of neutrophils. The mouse and the associated work has been published in the journal Nature Communications. 
URL http://www.nature.com/ncomms/2015/150416/ncomms7818/full/ncomms7818.html
 
Description Brain-Liver communication 
Organisation University of Oxford
Department Department of Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution My team helped with the anlysis of NF-kB activity in the liver and the brain. I was also a major contributor to planning of the experiments and writing of the paper.
Collaborator Contribution This collaboration enabled us to assess the role of IKK/NF-kB system in the communication between inflammatory signals between the brain and liver. This work was published in 2008. It also enabled me to learn about the in vivo imaging system, IVIS, which contributed to a succesful Wellcome Trust equipment grant to fund an IVIS in Newcastle.
Impact Pubmed ID 18344913.
Start Year 2006
 
Description Epigenetic control of fibrosis 
Organisation University of Southern California
Country United States 
Sector Academic/University 
PI Contribution My team have carried out a wide number of studies that reveal a pivotal role for the epigentic protein MeCP2 and its regulators in the control of NF-kB and liver fibrosis.
Collaborator Contribution A new area of research has been developed investigating the epigenetic control of liver fibrosis that has so far resulted in an NIAAA (NIH) R21 award plus two peer review publications.
Impact PMID: 16763620 and PMID: 19843474.
Start Year 2006
 
Description Sox9 and liver fibrosis 
Organisation University of Southampton
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution My team helped analyse the role of Sox9 in fibrosis by analysis of pathology slides and provision of materials from primary hepatic stellate cells and models of liver disease.
Impact PMID: 18296708
Start Year 2007
 
Description BBC Look North News Item 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A televised interview based in my laboratory which was transmitted on breakfast and lunchtime issues of BBC Look North on July 1st 2009.

Substantial public interest in our research (encouraging e mails, telephone calls etc) Plus a very good response to a call for patients in the North East with non-alcoholic fatty liver disease to be considered for a clinical trial supported by an EME.
Year(s) Of Engagement Activity 2009
 
Description British Liver Trust News Bulletin 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Following publication of key papers and news items on the BBC (Look North) and national + local newspapers the British Liver Trust (BLT) asked me to prepare a lay summary of our research discoveries on NF-kB and fibrosis reversion for their bulletin (issue 16).

The BLT provided a support letter for an application by Prof Chris Day and myself for a succesful NIHR/MRC EME application for a clinical trial to determine if the angiotensin receptor blocker Losartan can promote regression of fibrosis in patients with non-alcoholic fatty liver disease.
Year(s) Of Engagement Activity 2009
 
Description Leading Edge 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Schools
Results and Impact 6 pupils each year were brought into the laboratory to undertake a research project and then presented their work at local and national schools meetings and competitions.

The activity engaged pupils directly in research, teaching them experimentation, data collection and presentation in oral and written formats.
Year(s) Of Engagement Activity 2010,2011,2012
 
Description New Scientist 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Following a press conference organised by Newcastle University I helped the New Scientist prepare an article liver disease that highlighted the emerging problem of dietary-related liver disease. The article described a great deal of our work on reversal of liver disease and fibrosis.

Substantive interest from the general public with letters of support and many requests for further information and advice on liver disease and healthy lifestyle.
Year(s) Of Engagement Activity 2007
 
Description Research presentation to Liver North 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact LiverNorth is a well organised and well informed patient-ran charity that provides funding for liver disease resesrch and patient support in the North East of England. They have monthly meetings at which a basic or clinical research presenntation is provided by either a physician, scientist or surgeon. I presented the work my team has been carrying out on reversal of liver fibrosis.

Support letters from LiverNorth were provided for successful grant applications to the European Union and the MRC.
Year(s) Of Engagement Activity 2007