The Role of Tissue-resident Hepatic Macrophages in the Resolution of Chronic Liver Injury

Liver cirrhosis is the result of chronic liver damage from a variety of causes and is characterised by progressive scarring which eventually causes liver failure. Cirrhosis has been highlighted by the UK government as one of the "Big Five" causes of premature death and, worryingly, the rates of death continue to increase (over 400% since 1970) despite simultaneous reductions in death due to other common causes such as heart disease or cancer. Current treatments are unsatisfactory with options limited to removal of the underlying cause and liver transplantation for a select few. However, there is now evidence in virtually all liver diseases that even advanced scarring can at least partially resolve. Hence, by studying the mechanisms controlling this scar resolution, we could potentially develop badly-needed new treatments to improve cirrhosis and prevent liver failure and premature death.

Macrophages are cells of the immune system with numerous subtypes and a wide range of biological functions. They reside in all organs of the body and can also be derived from blood. Macrophages are essential for the removal of scar tissue in the liver and other organs. Thus, by understanding the biology of these scar resolving macrophages, we could potentially enhance their number and/or activity, with a beneficial effect on organ function. However, in order to target the appropriate population and limit side effects from potential treatments, it is imperative that we have a full understanding of the function of differing macrophage populations. One of the main distinguishing features of macrophage subpopulations in the liver is their origin. Resident liver macrophages are formed prior to birth, during embryological development, and maintain themselves into adulthood. Alternatively, in response to liver injury, cells (called monocytes) are recruited from the blood into the damaged liver and become a separate population of macrophages. My previous research identified that a population of blood-derived liver macrophages was responsible for the early phase of scar resolution following chronic liver damage. Liver injury also induces changes in resident liver macrophages, but the role of this population in scar resolution has not been studied. Thus, the objective of this proposed research will be to study the role of resident liver macrophages in scar resolution after chronic liver injury.

I will develop mouse models where I can identify resident liver macrophages and differentiate them from liver macrophages derived from blood. Being able to distinguish these cell types is essential to be able to attribute functions to either population. To determine the function of each macrophage population, I will use these mouse models to selectively remove either resident or blood-derived liver macrophages and assess the effect this has on the recovery from liver damage. To clarify how liver macrophage populations mediate their effects, I will isolate macrophage populations and analyse which genes they express. Similarly, I will isolate and analyse macrophages from human cirrhotic livers in patients undergoing liver transplantation, and compare them to the mouse macrophage results. This will enable me to identify pathways conserved between mouse and human liver disease, which is critical for development of potential treatments. Finally, I will use novel microscope technology to examine macrophages in real time within the livers of live mice following chronic injury. This will yield insights into the effects of macrophage localisation on function.

By completing this research, I will have characterised the role of resident liver macrophages in the regression of liver scarring after chronic injury. This will be a major advance in our understanding of mechanisms controlling the resolution of chronic liver damage, and will hopefully precede the development of targeted treatment strategies to improve liver function and reduce mortality.

Hepatic macrophages (MO) play a key role in liver fibrosis resolution. In order to target this therapeutically, a fuller understanding of MO heterogeneity and function is required. The role of embryologically-derived resident hepatic macrophages (RMO; Kupffer cells) in fibrosis resolution has not been defined. Preliminary data demonstrate that during resolution from murine chronic liver injury, hepatic RMO show changes in phenotype, topography and ontogeny to a mixed population of embryologically- (ED) and monocyte-derived (MD) RMO.

Aims:
1. To determine the ontogeny of RMO following chronic liver injury
2. To determine the function of RMO during liver fibrosis resolution
3. To perform a comparative systems biology analysis of murine and human hepatic macrophages
4. To analyse the hepatic RMO topographical niche during hepatic fibrosis resolution

Methodology: Novel Kupffer cell-specific cre transgenic mice will be used to fate map or selectively deplete hepatic EDRMO during liver fibrosis resolution. A haematopoietic reconstitution mouse model will be used to fate map or deplete hepatic MDMO during liver fibrosis resolution. Sequential intravital microscopy via an abdominal imaging window and laser capture microdissection will be used to characterise RMO motility, fate and topography. Murine and human hepatic RMO subpopulations will be isolated by FACS, with the transcriptome assessed and subjected to systems-biology based analysis.

Scientific and Medical Opportunities: By completing this research I will define the role of hepatic RMO in the resolution of chronic liver injury for the first time. Furthermore, by linking functional murine data to the phenotype of human hepatic macrophages using an unbiased systems-biology based approach, I hope to identify conserved pathways which can be targeted therapeutically to modulate macrophage function in vivo, promote liver fibrosis resolution and improve clinical outcomes for patients with cirrhosis.

Wider Scientific Community
This proposal will advance the knowledge of the role of macrophage subpopulations in the resolution of liver fibrosis. These data will be applicable to a broad range of scientific and medical disciplines. Tissue fibrosis is a major component of the pathophysiology of chronic diseases of the lung, kidney and cardiovascular system and has been estimated to contribute to up to 45% of deaths in the Western world. There is now evidence that common basic pathways underpin tissue fibrosis in multiple organs and fibrosis reversibility is widely-recognised with macrophages playing a key role. Hence, mechanisms discovered in the liver will be potentially applicable to a wide range of human diseases. Furthermore, macrophage responses are central to the pathogenesis of numerous other diseases including infection, inflammation and cancer. Thus, detailed characterisation of hepatic macrophage phenotypes will be of interest to macrophage biologists from a range of disciplines. The challenge to disseminate findings across such a broad field will be met through publication in high-impact peer-reviewed journals, but also presentation at local, national and international scientific meetings. In particular, I will not be limited to liver meetings but will also attend and present at general fibrosis and macrophage biology meetings such as Keystone, Cold Spring Harbor or Gordon conferences. I will also present data to medical researchers from other specialties both locally at the Queen's Medical Research Institute (QMRI) and nationally at meetings such as the Scottish Society for Experimental Medicine or Academy of Medical Sciences meeting for Clinician Scientists in Training.
Development of Novel Therapeutics
Another key facet of this proposed research is the comparative systems biology based analysis of murine and human hepatic macrophages with a view to identifying novel therapeutic targets to treat hepatic fibrosis. The QMRI is situated in close proximity to the Royal Infirmary of Edinburgh and the Scottish Liver Transplant Unit and is an ideal environment to allow prosecution of first-class basic science research, followed by translation into advances in patient care. Strong emphasis is also placed on exploitation of exciting research findings at the University of Edinburgh, with a dedicated subsidiary, Edinburgh Research and Innovation (http://www.research-innovation.ed.ac.uk/), offering management of technology transfer and company formation. Alternatively, we may utilise the expertise provided by MRC Technology to facilitate drug development opportunities. To engage directly with pharmaceutical and biotechnology companies, we may use established channels at Edinburgh Research and Innovation or present at national and international forums at which representatives are present.
Public Understanding of Science
I will aim to use this research programme to impact on the public understanding of science. This will be addressed in two ways: via the media and by direct interaction. The dedicated press offices of the University of Edinburgh and MRC will be used to disseminate research findings into the public domain. This will be supplemented by social media and podcasts, with media interviews where feasible. Direct interactions will be via participation at the Edinburgh Science festival (http://www.sciencefestival.co.uk), presentation at public lectures and engagement in open days. This will highlight the importance of basic medical research and hopefully inspire the next generation of biomedical scientists.
Economic Benefits
This work is internationally leading and will reinforce the MRC Centre for Inflammation Research and the University of Edinburgh as a world-renowned centre for liver macrophage biology. This will place us in an excellent position to compete for further funding including international grants such as those provided by EASL, which will have benefits for the University and the economy as a whole.