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Title: Investigating macrophage heterogeneity and function during acute liver injury
Author: Markose, Dyana
ISNI:       0000 0004 8510 3315
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Acetaminophen-induced liver injury (AILI) is the leading cause of acute liver failure (ALF) in Western Europe and USA. In ALF patients the intrinsic regenerative capacity of the liver is overwhelmed by massive hepatocellular death. Current treatment for AILI is limited to N-Acetylcysteine (NAC), the therapeutic potential of NAC decreases dramatically during the later stages of AILI and liver transplantation becomes the sole option. More potent therapies which promote liver regeneration which negates the need for transplantation is urgently required. Previous studies in both humans and animal models have highlighted monocytes and macrophages are key regulators of liver regeneration following AILI, due to their dynamic nature during AILI their therapeutic manipulation has proven to be challenging. In this thesis, using a murine model of AILI, I show specific hepatic leucocytes populated the liver at distinct phases of AILI, at the time of maximal liver regeneration macrophages represented the most dominant subset in the liver. Phenotypic characterisation of the three distinct macrophages populations: Kupffer cells (KCs), Ly6CHi monocytes and Ly6CLo monocyte derived macrophages (MDMs) indicated injury induced heterogeneity, particularly in the Ly6CLo MDMs. Droplet-based single cell RNA sequencing (scRNA-seq) was employed to interrogate monocyte and macrophage heterogeneity during liver regeneration, in an unbiased manner. Unsupervised clustering of mononuclear phagocytes from uninjured and post-AILI livers generated four distinct injury specific monocyte/MDM clusters. Gene ontology enrichment analysis indicated distinct functional roles for each cluster. Using newly identified marker genes, the heterogeneity within the Ly6CLo MDMs was validated in wild type and macrophage reporter mice. During maximal liver regeneration two distinct Ly6CLo MDMs populated the liver, they were defined as: CD63+ MHCII- and CD63- MHCII- MDMs. Identification of CD63+ MHCII- and CD63- MHCII- MDMs in the liver following CCl4 but not partial hepatectomy suggested it is an inflammation-induced phenotype. Experiments in CCR2 deficient mice indicated both macrophage subsets are a progeny of bone-marrow derived circulating monocytes. Localization of CD63+ macrophages to areas of resolving liver necrosis, adjacent to proliferating hepatocytes and their increased phagocytic capacity suggested a pro-repair role for CD63+ MHCII- MDMs. Additionally, scRNA-seq of circulating monocytes indicated AILI resulted in transcriptional pre-programming of circulating monocytes into an inflammatory phenotype, prior to their entry into the liver. These results support the notion that both circulating and hepatic monocytes and macrophages are highly dynamic and utilization of scRNA-seq in combination with traditional techniques such as flow cytometry and immunohistochemistry has facilitated the delineation of macrophage heterogeneity during liver regeneration. Further studies in mouse models and in humans are required to investigate the functional significance of these cells in vivo.
Supervisor: Henderson, Neil ; Pollard, Jeffrey Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: acetaminophen poisoning ; APAP poisoning ; macrophages ; liver failure ; mouse model ; scRNA-seq ; fluorescent macrophages