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Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists' reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer's "InForm" software package to semi-Automate the scoring of patient liver biopsies, and compare outputs to a pathologist's assessment. We examined a cohort of eleven acute hepatitis samples and three non-Alcoholic fatty liver disease (NAFLD) samples, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45 +ve inflammatory cells, areas of necrosis, fat and collagen deposition (p < 0.0001). Hepatitis samples were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r 2 = 0.8192, p < 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis.

The global prevalence of liver cancer is rapidly rising, mostly as a result of the amplified incidence rates of viral hepatitis, alcohol abuse and obesity in recent decades. Treatment options for liver cancer are remarkably limited with sorafenib being the gold standard for advanced, unresectable hepatocellular carcinoma but offering extremely limited improvement of survival time. The immune system is now recognised as a key regulator of cancer development through its ability to protect against infection and chronic inflammation, which promote cancer development, and eliminate tumour cells when present. However, the tolerogenic nature of the liver means that the immune response to infection, chronic inflammation and tumour cells within the hepatic environment is usually ineffective. Here we review the roles that immune cells and cytokines have in the development of the most common primary liver cancer, hepatocellular carcinoma (HCC). We then examine how the immune system may be subverted throughout the stages of HCC development, particularly with respect to immune inhibitory molecules, also known as immune checkpoints, such as programmed cell death protein-1, programmed cell death 1 ligand 1 and cytotoxic T lymphocyte antigen 4, which have become therapeutic targets. Finally, we assess preclinical and clinical studies where immune checkpoint inhibitors have been used to modify disease during the carcinogenic process. In conclusion, inhibitory molecule-based immunotherapy for HCC is in its infancy and further detailed research in relevant in vivo models is required before its full potential can be realised.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The global prevalence of liver cancer is rapidly rising, mostly as a result of the amplified incidence rates of viral hepatitis, alcohol abuse and obesity in recent decades. Treatment options for liver cancer are remarkably limited with sorafenib being the gold standard for advanced, unresectable hepatocellular carcinoma but offering extremely limited improvement of survival time. The immune system is now recognised as a key regulator of cancer development through its ability to protect against infection and chronic inflammation, which promote cancer development, and eliminate tumour cells when present. However, the tolerogenic nature of the liver means that the immune response to infection, chronic inflammation and tumour cells within the hepatic environment is usually ineffective. Here we review the roles that immune cells and cytokines have in the development of the most common primary liver cancer, hepatocellular carcinoma (HCC). We then examine how the immune system may be subverted throughout the stages of HCC development, particularly with respect to immune inhibitory molecules, also known as immune checkpoints, such as programmed cell death protein-1, programmed cell death 1 ligand 1 and cytotoxic T lymphocyte antigen 4, which have become therapeutic targets. Finally, we assess preclinical and clinical studies where immune checkpoint inhibitors have been used to modify disease during the carcinogenic process. In conclusion, inhibitory molecule-based immunotherapy for HCC is in its infancy and further detailed research in relevant in vivo models is required before its full potential can be realised.

© 2016 Elsevier Masson SASBackground & aims The availability of non-tumorigenic and tumorigenic liver progenitor cell (LPC) lines affords a method to screen putative anti-liver cancer agents to identify those that are selectively effective. To prove this principle we tested thalidomide and a range of its derivatives and compared them to lenalidomide and sorafenib, to assess their growth-inhibitory effects. Methods Cell growth, the mitotic and apoptotic index of cell cultures were measured using the Cellavista instrument (SynenTec) using commercially available reagents. Results Neither lenalidomide nor thalidomide (100 µM) affected tumorigenic LPCs but killed their non-tumorigenic counterparts. Sorafenib arrested growth in both cell types. All but two derivatives of thalidomide were ineffective; of the two effective derivatives, one (thalidomide C1) specifically affected the tumorigenic cell line (10 µM). Mitotic and apoptotic analyses revealed that thalidomide C1 induced apoptotic cell death and not mitotic arrest. Conclusions This study shows that screens incorporating non-tumorigenic and tumorigenic liver cell lines are a sound approach to identify agents that are effective and selective. A high throughput instrument such as the Cellavista affords robust and reproducible objective measurements with a large number of replicates that are reliable. These experiments show that neither lenalidomide nor thalidomide are potentially useful for anti-liver cancer therapy as they kill non-tumorigenic liver cells and not their tumorigenic counterparts. Sorafenib in contrast, is highly effective, but not selective. One tested thalidomide derivative has potential as an anti-tumor drug since it induced growth arrest; and importantly, it selectively induced apoptotic cell death only in tumorigenic liver progenitor cells.

Liver progenitor cells (LPCs) are necessary for repair in chronic liver disease because the remaining hepatocytes cannot replicate. However, LPC numbers also correlate with disease severity and hepatocellular carcinoma risk. Thus, the progenitor cell response in diseased liver may be regulated to optimize liver regeneration and minimize the likelihood of tumorigenesis. How this is achieved is currently unknown. Human and mouse diseased liver contain two subpopulations of macrophages with different ontogenetic origins: prenatal yolk sac-derived Kupffer cells and peripheral blood monocyte-derived macrophages. We examined the individual role(s) of Kupffer cells and monocyte-derived macrophages in the induction of LPC proliferation using clodronate liposome deletion of Kupffer cells and adoptive transfer of monocytes, respectively, in the choline-deficient, ethionine-supplemented diet model of liver injury and regeneration. Clodronate liposome treatment reduced initial liver monocyte numbers together with the induction of injury and LPC proliferation. Adoptive transfer of monocytes increased the induction of liver injury, LPC proliferation, and tumor necrosis factor-a production. Conclusion: Kupffer cells control the initial accumulation of monocyte-derived macrophages. These infiltrating monocytes are in turn responsible for the induction of liver injury, the increase in tumor necrosis factor-a, and the subsequent proliferation of LPCs.

Background & Aims: Although a strong association between liver progenitor cells (LPCs) and inflammation exists in many chronic liver diseases, the exact role of the immune system in LPC-mediated hepatic regeneration remains unclear. A number of pro-inflammatory factors were identified in cytokine knockout mice in which the LPC response was attenuated but neither the mechanism nor the producing cells are known. Methods: To identify the critical immune cells and cytokines required in the LPC response, we compared two diet-induced models of liver injury with two recently established transgenic models of immune-mediated hepatitis. Results: Despite severe inflammation being observed in all models, the generation of LPCs was highly dependent on the cause and kinetics of liver damage. The LPC response was associated with an increase of macrophages and CD8+ T cells but not natural killer cells. T cell-deficient mice were able to mount a LPC response, albeit delayed, suggesting that T cells are not essential. Mice mounting an LPC response showed elevated numbers of Kupffer cells and invading CX3CR1 highCCR2high macrophages secreting persistent high levels of tumour necrosis factor alpha (TNFa), a major cytokine involved in the LPC response. Conclusions: Liver macrophages are an important determinant of LPC expansion during liver regeneration in models of diet- and immune-mediated liver injury. Invading macrophages in particular provide pro-mitogenic cytokines such as TNFa that underpin the process. LPC themselves are a source of chemokines (CCL2, CX3CL1) that attract infiltrating macrophages. © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

In macrophages, tyrosine phosphorylation regulates many signalling pathways leading to growth, differentiation, activation, phagocytosis and adhesion. Protein tyrosine phosphatases (PTPs) represent a biochemical counterbalance to the activity of protein tyrosine kinases, thus regulating the dynamic phosphorylation state of a cell. CD148 is a receptor PTP that is highly expressed in macrophages and is further regulated by pro-inflammatory stimuli. CD148 is normally localised to the plasma membrane of macrophages. Following stimulation with CSF-1 or LPS, there was a re-distribution and concentration of CD148 in areas of membrane ruffling. Treatment of macrophages with anti-CD148 monoclonal antibody inhibited CSF-1-induced macrophage spreading, cytoskeletal re-arrangements and chemotaxis, without affecting cell survival. There were no detectable effects on the CSF-1 receptor-akt signalling pathway. These results are consistent with the hypothesis that CD148 is a regulator of macrophage activity. © 2009 Elsevier Inc. All rights reserved.

OBJECTIVE-: Oxidized low-density lipoprotein (oxLDL) induces survival of colony stimulating factor-1 (CSF-1)-dependent macrophages in vitro. Because atherosclerotic lesion-associated macrophages take up large amounts of glucose, we investigated whether, and how, oxLDL promotes glucose uptake and how glucose metabolism regulates oxLDL-induced macrophage survival. METHODS AND RESULTS-: OxLDL-induced macrophage survival required glucose metabolism. OxLDL stimulated 2 phases of glucose uptake, namely acute and chronic, which required PI3K but not MEK1/2 activity. PI3K appeared to regulate glucose transport via glucose transporter affinity and/or mobilization. OxLDL also maintained levels of the prosurvival proteins, Bcl-2 and Bcl-xL, after CSF-1 had been removed through a combination of mechanisms including transcription, translation, and protein stabilization. Significantly, inhibition of glucose metabolism reduced Bcl-2 and Bcl-xL protein levels. MEK1/2 and PI3K activities were also required for oxLDL-induced Bcl-2 and Bcl-xL mRNA upregulation. CONCLUSIONS-: These results suggest that oxLDL enhances macrophage survival in the absence of CSF-1 by inducing PI3K-dependent glucose uptake, which is metabolized to maintain Bcl-2 and Bcl-xL protein levels. © 2009 American Heart Association, Inc.

Uptake by macrophages forms an important part of the mode of action of particulate adjuvants such as oil-in-water emulsions and alum. We have found previously that such adjuvants promote macrophage survival and suggested that this response may contribute to their efficacy. To explore this adjuvant activity further, we have investigated whether oil-in-water emulsion stimulates glucose uptake in macrophages and whether such uptake is relevant to the promotion of survival. We found that oil-in-water emulsion stimulated glucose uptake in a biphasic manner. The first acute phase was independent of mRNA and protein synthesis but appeared to require PI3K activity. In contrast, the second chronic phase was dependent on mRNA and protein synthesis. Importantly, the second phase of glucose uptake required MEK1/2 as well as PI3K activity, indicating that the MEK1/2 pathway can also contribute to cellular glucose uptake. The increased glucose transporter 1 expression during the second phase and long-term survival also appeared to be dependent on PI3K and MEK1/2 signaling pathways. Metabolism of the glucose was required for the emulsion-stimulated survival as well as the increase of prosurvival Bcl-2 transcript levels and maintenance of Bcl-2 protein expression. As transgenic overexpression of Bcl-2 enhances the survival of macrophages in the absence of growth factor, the glycolytic control of Bcl-2 levels may play a central role in emulsionstimulated macrophage survival. Enhanced glucose uptake by macrophages may therefore be critical to the action of particulate adjuvants. © Society for Leukocyte Biology.

In chronic inflammatory lesions macrophages are abundant and adapt to the low oxygen concentrations often present there. In low oxygen some cell types die by apoptosis, as reported for macrophage cell lines, while others survive better as they shift their metabolism to anaerobic glycolysis. It was found here that hypoxia prolongs the survival of murine bone marrow-derived macrophages, either in the absence or presence of low CSF-1 (M-CSF) concentrations. Although Akt activity increased in bone marrow-derived macrophages in the low oxygen conditions, the levels of both anti- and proapoptotic Bcl-2 family members decreased. Glycolysis was enhanced as judged by increased glucose uptake, glucose transporter expression, lactate dehydrogenase mRNA expression, and lactate secretion. Human monocytes responded similarly to low oxygen, and a number of genes associated with glycolysis were shown by microarray analysis and quantitative PCR to be up-regulated. Interestingly, human monocytederived macrophages showed evidence of enhanced glycolysis even under aerobic conditions. It is proposed that certain monocyte/macrophage populations survive better under conditions of low oxygen, thereby contributing to their increased numbers at sites of chronic inflammation and tumors; it is also proposed that as macrophages differentiate from monocytes they begin to adopt a glycolytic metabolism allowing them to adapt readily when exposed to low oxygen conditions. Copyright © 2009 by The American Association of Immunologists, Inc.

Colony stimulating factor-1 (CSF-1)-dependent macrophages play crucial roles in the development and progression of several pathological conditions including atherosclerosis and breast cancer metastasis. Macrophages in both of these pathologies take up increased amounts of glucose. Since we had previously shown that CSF-1 stimulates glucose uptake by macrophages, we have now investigated whether glucose metabolism is required for the survival of CSF-1-dependent macrophages as well as examined the mechanism by which CSF-1 stimulates glucose uptake. Importantly, we found that CSF-1-induced macrophage survival required metabolism of the glucose taken up in response to CSF-1 stimulation. Kinetic studies showed that CSF-1 stimulated an increase in the number of glucose transporters at the plasma membrane, including Glut1. The uptake of glucose induced by CSF-1 required intact PI3K and PLC signalling pathways, as well as the downstream effectors Akt and PKC, together with a dynamic actin cytoskeleton. Expression of constitutively active Akt partially restored glucose uptake and macrophage survival in the absence of CSF-1, suggesting that Akt is necessary but not sufficient for optimal glucose uptake and macrophage survival. Taken together, these results suggest that CSF-1 regulates macrophage survival, in part, by stimulating glucose uptake via Glut1, and PI3K and PLC signalling pathways. © 2009 Elsevier Inc.

Elsegood, C. L., Tirnitz-Parker, J. E., Olynyk, J. K., & Yeoh, G. C. (2017). Immune checkpoint inhibition: prospects for prevention and therapy of hepatocellular carcinoma. Clinical & Translational Immunology, 6(11), e161. Available here

Gogoi-Tiwari, J., Köhn-Gaone, J., Giles, C., Schmidt-Arras, D., Gratte, F. D., Elsegood, C. L., ... & Tirnitz-Parker, J. E. (2017). The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury. Journal of visualized experiments: JoVE, (128). Available here.