roles of interferon α and γ in the hepatic progenitor ... · v the following document describes...

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Roles of Interferon α and γ in the

Hepatic Progenitor (Oval) Cell

Response

Rebecca Lim BSc (Hons), Dip Biotech

Submitted for Doctor of Philosophy (Medicine and Pharmacology)

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Abstract Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem

cells capable of regenerating the liver during chronic liver injury and also as targets of

malignant transformation. Similar markers are expressed by hepatocellular carcinoma

(HCC) and HPC, and a precursor-product relationship is well established. This thesis

focuses on the ways in which the HPC population can be controlled under circumstances of

chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced.

The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy

on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide.

Approximately 80% of infections progress to chronicity, which places the individuals at

greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a

combination of pegylated IFNα and ribavirin. IFNα therapy has been shown to reduce the

risk of HCC development in chronic hepatitis C patients regardless of whether the patients

achieve sustained virological response (SVR). To date there has been no study to determine

the impact that IFNα has on the HPC population. The first experimental chapter of this

thesis revealed that IFNα therapy resulted in a reduction in HPC numbers (average

reduction of 50%) in 13 out of 16 patients (p<0.01), most of whom did not achieve SVR. In

vitro studies showed that IFNα exerts anti-proliferative and pro-apoptotic effects on two

well characterized murine HPC cell lines - PIL-2 and PIL-4. Additionally, IFNα treatment

induced differentiation of these cells causing expression of mature hepatocyte and biliary

markers.

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Studies utilizing mice fed a choline deficient, ethionine supplemented (CDE) diet

confirmed that IFNα treatment reduced HPC numbers in vivo. Following 14 days of

treatment with pegylated IFNα, numbers of A6-, MPK- and c-kit-positive HPC were

significantly reduced (p<0.05). This was accompanied by a slight increase in HPC

apoptosis and a concurrent reduction in the number of activated hepatic stellate cells

(HSC), as well as a significant decrease in expression of LTβ and IFNγ (p<0.05). In

summary, these results suggest that IFNα therapy is useful treatment for patients with

chronic hepatitis C even if they do not attain SVR. IFNα therapy could effectively reduce

the risk of HCC development in these patients by bringing the HPC population under

control through its effects on differentiation, apoptosis and proliferation.

The second aim of this thesis (Chapters 6 to 7) was to determine the effects of IFNγ on

HPC. This cytokine has been previously reported to be an essential mediator of the HPC

response. With an increasing interest in exploitation of IFNγ for its antagonistic effects on

transforming growth factor β (TGFβ) in treatment of fibrosis, we sought to evaluate its

effects on HPC. The results were surprising. While IFNγ exerted a pro-apoptotic and anti-

proliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days

increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response,

with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were

observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime

the liver for the HPC response by recruiting inflammatory cells and altering the hepatic

cytokine profile, both of which may facilitate an increased HPC response. Numbers of

activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the

increased fibrosis seen in these animals. This data contradicts the current experimental use

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of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC

proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte

damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated

HSC further supports the view that there is a close relationship between the two cell types,

and thus, a link between the HPC response and fibrosis.

In conclusion, findings documented in this thesis suggest that administration of IFNα and

IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in

chronic hepatitis C patients by bringing the HPC population under control. In contrast,

IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage,

illustrating the need to approach this method of fibrosis treatment with caution.

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The following document describes the percentage contribution made by each author to the published work contained within this PhD thesis. Lim, R., Knight, B., Patel, K., McHutchison, J., Yeoh, G. and Olynyk, J. (2006) Antiproliferative effects of Interferon Alpha on Hepatic Progenitor Cells In Vitro and In vivo. Hepatology. (43):1074-1083. Authors and distribution of work: Rebecca Lim 55% Belinda Knight 20% Keyur Patel 5% John McHutchinson 5% George Yeoh 5% John Olynyk 10% ***Manuscript submitted to Hepatology Lim, R., Knight, B., Yeoh, G. and Olynyk, J. (2006) Interferon gamma increase numbers of hepatic progenitor cells and exacerbates fibrosis in the CDE model. Authors and distribution of work: Rebecca Lim 40% Belinda Knight 40% George Yeoh 10% John Olynyk 10%

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Table of Contents Acknowledgements ................................................................................................................ x Publications ........................................................................................................................... xi Poster Presentations .............................................................................................................. xi Oral Presentations ................................................................................................................. xi List of Abbreviations ........................................................................................................... xii List of Figures ..................................................................................................................... viii List of Tables ..................................................................................................................... xvii 1. General Introduction ....................................................................................................... 1

1.1. Anatomy, Physiology and Vasculature ....................................................................... 1 1.2. Liver Development ..................................................................................................... 3 1.3. Liver Regeneration...................................................................................................... 4 1.4. Cellular Components of the Liver............................................................................... 6

1.4.1. Kupffer Cells........................................................................................................ 6 1.4.2. Hepatic Stellate Cells ........................................................................................... 7 1.4.3. Hepatic Progenitor Cells ...................................................................................... 8

1.5. Hepatic Progenitor Cell Activation in Chronic Liver Diseases ................................ 10 1.5.1. Chronic Hepatitis C............................................................................................ 10 1.5.2. Hepatic Progenitor Cells as Targets of Malignant Transformation ................... 13

1.6. Models of Progenitor Cell Activation....................................................................... 16 1.7. Hepatic Progenitor Cell Lines................................................................................... 19 1.8. Inflammatory Cytokines and the Progenitor Cell Response ..................................... 21

1.8.1. Tumor Necrosis Factor-α ................................................................................... 22 1.8.2. Lymphotoxin-β................................................................................................... 22 1.8.3. Interleukin-6....................................................................................................... 23 1.8.4. Oncostatin M...................................................................................................... 23

1.9. Interferon α................................................................................................................ 24 1.9.1. Interferon Signaling Pathways ........................................................................... 24 1.9.2. Effects on Tumor Cell Lines .............................................................................. 26

1.10. Interferon γ .............................................................................................................. 27 1.10.1. Interferon γ in the Treatment of Fibrosis ......................................................... 29 1.10.2. Recent Interest in Role of Interferon γ in Hepatic Progenitor Cell Response . 30

1.11. Aims of Project ....................................................................................................... 31 2. General Materials and Methods ................................................................................... 32

2.1. Immunohistochemical Methods................................................................................ 32 2.1.1. Tissue Fixation, Sectioning and Deparaffinization............................................ 32 2.1.2. Antigen Retrieval ............................................................................................... 32 2.1.3. Tissue Section Mounting and Microscopy......................................................... 33

2.2. Cell Culture Techniques............................................................................................ 33 2.2.1. Maintenance of Cell Cultures ............................................................................ 33 2.2.2. Serum Starvation................................................................................................ 34

2.3. Molecular Techniques............................................................................................... 34 2.3.1. RNA Isolation from Cells .................................................................................. 34 2.3.2. RNA Isolation From Liver Tissue ..................................................................... 35 2.3.3. DNAse Treatment of RNA ................................................................................ 36

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2.3.4. cDNA Synthesis ................................................................................................. 36 2.3.5. Cloning Gene Products ...................................................................................... 37 2.3.6. Plasmid screening .............................................................................................. 38

2.4. Terminal dUTP-nick End Labeling (TUNEL) Assay ............................................... 39 2.5. Animal Studies .......................................................................................................... 40

2.5.1. CDE Feeding...................................................................................................... 40 2.5.2. Euthanasia .......................................................................................................... 40

3. Interferon α Decreases Numbers of Hepatic Progenitor Cells in Patients with Chronic Hepatitis C ........................................................................................................... 41

3.1. Introduction............................................................................................................... 41 3.2. Materials and Methods.............................................................................................. 45

3.2.1. Tissue Samples................................................................................................... 45 3.2.2. Immunohistochemical Staining of Hepatic Progenitor Cells and Inflammatory Infiltrate........................................................................................................................ 47 3.2.3. Statistical Analysis ............................................................................................. 48

3.3. Results ....................................................................................................................... 49 3.3.1. Interferon α Treatment Reduced Hepatic Progenitor Cell Numbers as Demonstrated by Immunohistochemical Staining with c-kit, CK19 and π-GST ........ 49 3.3.2. Interferon-α Treatment Did Not Affect Inflammatory Infiltration.................... 51

3.4. Discussion ................................................................................................................. 52 4. Effect of Interferon α on Proliferation, Differentiation and Apoptosis of Hepatic Progenitor Cells in vitro ..................................................................................................... 56

4.1. Introduction............................................................................................................... 56 4.2. Materials and Methods.............................................................................................. 58

4.2.1. Stimulation of PIL-2 and PIL-4 with Interferon α............................................. 58 4.2.2. Determining the Effect of Interferon α on Mitochondrial Activity Using MTT Assay............................................................................................................................ 59 4.2.3. Determining the Effect of Interferon α on Proliferation using Proliferating Cell Nuclear Antigen Staining............................................................................................. 60 4.2.4. Determining the Effect of Interferon α on Cyclin D1 Expression..................... 61 4.2.5. Detecting Apoptotic Cells Using Terminal dUTP Nick End Labeling (TUNEL) Assay............................................................................................................................ 62 4.2.6. Determining Changes in Differentiation Status Using Quantitative PCR ......... 62

4.3. Results ....................................................................................................................... 64 4.3.1. Interferon α Inhibits Proliferation in Both PIL-2 and PIL-4.............................. 64 4.3.2. Interferon α Reduces Cyclin D1 Expression in PIL-2 But Not PIL-4 ............... 66 4.3.3. Interferon α Causes Apoptosis in Both PIL-2 and PIL-4 .................................. 67 4.3.4. Interferon α Causes PIL-2 and PIL-4 Cells to Differentiate.............................. 68 4.3.5. Interferon α Did Not Affect the Morphology of PIL-2 and PIL-4 .................... 71

4.3. Discussion ................................................................................................................. 73

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5. Effects of Interferon α on Hepatic Progenitor Cells in vivo ....................................... 75 5.1. Introduction............................................................................................................... 75

5.2.1. Animal Studies ................................................................................................... 77 5.2.2. Immunohistochemical Staining for A6, M2-Pyruvate Kinase (MPK) and c-kit 77 5.2.3. Determining Changes in Proliferative Status Using PCNA Staining ................ 78 5.2.4. Determining Changes in Apoptotic Status Using TUNEL Staining.................. 78 5.2.5. RNA Isolation from Mouse Liver Tissues ......................................................... 78 5.2.6. Determing Effect of Interferon α on Cytokine Profile ...................................... 79 5.2.7. Histological Grading of Fibrosis........................................................................ 80 5.2.8. Immunohistochemical Staining for Activated Hepatic Stellate Cells................ 80 5.2.9. Immunohistochemical Staining for Phosphorylated STAT-3............................ 81

5.3. Results ....................................................................................................................... 82 5.3.1. Interferon α Reduces Numbers of Hepatic Progenitor Cells in the CDE Mouse Model ........................................................................................................................... 82 5.3.2. Interferon α Exerts a Differential Effect on Proliferation of Hepatic Progenitor Cells and Hepatocytes .................................................................................................. 85 5.3.3. Interferon α Treatment Induces Apoptosis ........................................................ 86 5.3.4. Interferon α Treatment Changes Cytokine Profile of CDE-fed Mice ............... 88 5.3.5. Interferon α Does Not Significantly Affect Fibrotic Status of CDE-fed Mice.. 90 5.3.6. Interferon α Treatment Significantly Reduces Numbers of Activated Hepatic Stellate Cells ................................................................................................................ 91 5.3.7. Phosphorylation of STAT-3 as a Molecular Mechanism of Interferon α Action...................................................................................................................................... 92

5.4. Discussion ................................................................................................................. 94 6. Effects of Interferon γ on Hepatic Progenitor Cells in vitro ...................................... 99

6.1. Introduction............................................................................................................... 99 6.2. Methods................................................................................................................... 101

6.2.1. Detecting the Presence of α and β Chains of the Interferon γ Receptor .......... 101 6.2.2. Measuring of the Effect of IFNγ on Proliferation of Hepatic Progenitor Cells Using MTT Assay and BrdU Incorporation .............................................................. 102 6.2.3. Measuring the Effect of IFNγ on Apoptosis Using TUNEL Assay................. 103 6.2.4. Determining the Effect of Interferon γ on Differentiation Status of the Hepatic Progenitor Cells.......................................................................................................... 104 6.2.5. Primary Hepatic Progenitor Cell Isolation....................................................... 104

6.3. Results ..................................................................................................................... 106 6.3.1. Both α and β Chains of IFNγ Receptor are Expressed by PIL-2 and PIL-4 .... 106 6.3.2. Interferon γ Inhibits Proliferation of PIL-2 and PIL-4 in vitro ........................ 107 6.3.3. Interferon γ Induced Apoptosis in PIL-2 But Not PIL-4 ................................. 108 6.3.4. Treatment of PIL-2 and PIL-4 with Interferon γ Changed Expression of Hepatic Maturation Markers.................................................................................................... 109 6.3.5. Confirmation of Cell Line Data in Primary Progenitor Cells .......................... 112

6.4. Discussion ............................................................................................................... 113

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7. Effects of Interferon γ on Hepatic Progenitor Cells in vivo...................................... 116 7.1. Introduction............................................................................................................. 116 7.2. Methods................................................................................................................... 117

7.2.1. Treating CDE-fed mice with Interferon γ ........................................................ 117 7.2.2. Feeding the CDE Diet ...................................................................................... 117 7.2.3. Immunohistochemical Staining for A6, M2-Pyruvate Kinase (MPK) and Stem Cell Factor Receptor (c-kit) ....................................................................................... 118 7.2.4. Sirius Red Staining for Detecting Change in Fibrosis ..................................... 118 7.2.5. Determining Change in Apoptotic Status Using TUNEL Staining ................. 118 7.2.6. Determining Changes in Proliferative Status Using Cytokeratin/Ki67 Double Immunohistochemical Staining.................................................................................. 119 7.2.7. Detection of Inflammatory Influx Using CD45 Immunohistochemical Staining.................................................................................................................................... 120 7.2.8. Analysis of Hepatic Cytokine Profile .............................................................. 120

7.3. Results ..................................................................................................................... 121 7.3.1. Interferon γ Increased Numbers of MPK-, A6- and c-kit-positive Hepatic Progenitor Cells in CDE-fed Mice............................................................................. 121 7.3.2. Interferon γ Exerted a Differential Effect on Apoptosis of Hepatic Progenitor Cells and Hepatocytes in vivo .................................................................................... 124 7.3.3. Interferon γ Exacerbated Inflammation and Fibrosis in CDE-fed Mice .......... 126 7.3.4. Interferon γ Induced Changes in the Hepatic Cytokine Profile ....................... 128 7.3.5. Interferon γ Invoked Inflammatory Cell Infiltration and Exacerbated Hepatocyte Damage During Early Stages of CDE-feeding .......................................................... 130

7.4. Discussion ................................................................................................................... 142 8. General Discussion....................................................................................................... 147

8.1. Introduction............................................................................................................. 147 8.2. Hepatic Progenitor Cells and Cancer ...................................................................... 147 8.2. Interferon γ Modulates the Hepatic Progenitor Cell Response............................... 149 8.3. Concluding Remarks............................................................................................... 152

Bibliography ..................................................................................................................... 155

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Acknowledgements It is no real secret that a PhD thesis is never the work of a single individual. While this thesis has my name printed on it, there are many others who contributed to this piece of work in one way or another. Some gave technical advice when they found me wearing out the carpet in my office with my endless pacing, whilst ranting and raving and swearing to give up science altogether. Others proof-read my chapters and manuscripts; covering them with red-inked comments, doodles and the occasional coffee stain. More helped out just with a kind word or two, a pat on the back and reassuring words. This thesis is for all of you. To my supervisors, George and John – thank you for believing in me. Thank you for guiding me through what can only be described as a trying time for all involved. Standing on the shoulders of giants has made my PhD years pass quickly by. To my family – Thank you for your understanding and endless support. Thank you for being proud of me, and knowing that this was something that I had to do. To my fellow labmates at Fremantle Hospital, both past and present: Firstly, the woman who was more than a labmate, who walked me through the steps of cell biology and taught me how to write my first paper, Belinda Knight. Leon Brownrigg and Borut Klopcic – How could anyone survive a PhD without someone to have coffee with? The Iron Group Women – Debbie Trinder, Anita Chua & Carly Herbison – What great company you have been! Jane Allan – the best lab manager and all around top woman ever! To my friends at Budokan Academy – Marcus Phung, Michael Han, Michele Ong and Chris Dowling a.k.a. the Chinese Mafia (with our mandatory Australian intake). Thank you for the laughs and the great times both in and outside the dojo. To the Lawrence family: Ray and Norma for taking me in into their lives and into their home. Russell and Gavin for being the two older brothers I never had! To Mohan, without whom I would have given up on this crazed idea of doing a PhD a l o n g time ago. Thank you for letting me throw my tantrums, for coming to the lab with me in the middle of the night so that I could run ‘just one more experiment’, for watching Thunder so I could attend a conference. Thank you. To Marcus Lee-Steere who made me sit down and do all the necessary revisions so I could finally graduate!!!

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Publications Lim, R., Knight, B., Patel, K., McHutchison, J., Yeoh, G. and Olynyk, J. (2006) Antiproliferative effects of Interferon Alpha on Hepatic Progenitor Cells In Vitro and In vivo. Hepatology. (43):1074-1083. ***Manuscript submitted to Hepatology Lim, R., Knight, B., Yeoh, G. and Olynyk, J. (2006) Interferon gamma increase numbers of hepatic progenitor cells and exacerbates fibrosis in the CDE model.

Poster Presentations Effect of Interferon-alpha on hepatic progenitor cells in patients with chronic hepatitis C. Lorne Cancer Conference, Lorne, Victoria. (2006) Interferon gamma exacerbates fibrosis and increases hepatic progenitor cell numbers in CDE-fed mice. Digestive Disease Week, Los Angeles, USA. (2006).

Oral Presentations Effect of antiviral therapy on hepatic progenitor cells in patients with chronic hepatitis C and risk for hepatocellular carcinoma. Australian Society of Medical Researchers Annual Symposium. Perth, Western Australia (2004) Effect of interferon-alpha on hepatic progenitor cells in patients with chronic hepatitis C. Australian Gastroenterology Week. Brisbane, Queensland. (2004) Antiviral treatment of chronic hepatitis C: Implications on hepatocarcinogenesis. School of Medicine and Pharmacology Research Showcase. Perth, Western Australia. (2005) Effect of antiviral treatment on hepatic progenitor cells in chronic hepatitis C patients and its implications on liver cancer. Combined Biological Sciences Meeting. Scarborough, Western Australia. (2005)

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List of Abbreviations 2-AAF 2-acetylaminofluorene 3’-Me-DAB 3’-methyl diaminobenzene π-GST π-glutathione-S-transferase BMEL cells Bipotential murine embryonic liver cells BMOL cells Bipotential murine oval liver cells CDE diet Choline deficient ethione supplemented diet CK Cytokeratin c-kit Stem cell factor receptor DEN Diethylnitrosamine DMN Dimethylnitrosamine ECM Extracellular matrix GGT γ-glutamyl transferase HCC Hepatocellular carcinoma HCV Hepatitis C virus hFLMPC Human fetal liver multipotential cells HPC Hepatic progenitor cells HSC Hepatic stellate cells IFNα Interferon α IFNγ Interferon γ IL-6 Interleukin-6 Jak Janus kinase LTβ Lymphotoxin β M2PK Muscle isoenzyme 2 pyruvate kinase NF-κB Nuclear factor-κ B OSM Oncostatin M PDGF Platelet derived growth factor PH Partial hepatectomy PIL p53-null immortalized liver cells ROS Reactive oxygen species SCF Stem cell factor STAT Signal transducers and activators of transcription TGFβ Transforming growth factor β TNFα Tumor necrosis factor α

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List of Figures Figure 1.1. Diagrammatic representation of a liver acinus 2.................................................. 2 Figure 1.2. A representative diagram of the structure of the hepatitis C virus genome. The genome carries an open reading frame (ORF) encoding a polypeptide precursor of 3010 amino acids. Its translation is directed via a ~340 bp nucleotide long 5’ non-translated region (NTR), which functions as an internal ribosome entry site. It allows for direct binding of ribosomes in close proximity to the start codon of the ORF. The polypeptide is then cleaved to form 10 products. The structural proteins, C, E1 and E2 at the first third from the N-terminal, and the non-structural replicative proteins, N2-5, located further. Putative functions of these proteins are stated above. ......................................................... 12 Figure 1.3. Schematic diagram showing the molecular pathways used for interferon α signaling 190. ......................................................................................................................... 25 Figure 1.4. Schematic diagram of IFNγ signaling via the JAK-STAT pathway 203. ........... 28 Figure 3.1. Change in HPC numbers determined by immunohistochemical staining. Following treatment, c-kit-positive cells decreased by 50% in 13 out of 16 patients (*p<0.05, A). However it did not cause any significant changes in the CK19 (B) or π−GST-(C) positive cells. ................................................................................................................. 50 Figure 3.2. Identification of inflammatory cells using LCA immunohistochemical staining. Treatment with IFNα did not appear to change inflammatory status of chronic hepatitis C patients. ................................................................................................................................ 51 Figure 4.1. MTT-based proliferation assay. Effect of IFNα (250 IU/mL) on proliferation of PIL-2 cells (A) and PIL-4 cells (B) after 24 hours in culture. ............................................. 64 Figure 4.2. Treatment with IFNα changes proliferation status of PIL-2 and PIL-4 cells. Treatment with IFNα caused a significant decrease in PCNA-positive PIL-2 cells (A**p<0.001). Representative photographs of PCNA staining of PIL-2 cells grown in control media and media supplemented with 250 IU/mL IFNα are shown in B and C respectively. Similarly, IFNα treatment resulted in a decrease in PCNA-positive PIL-4 cells (D. **p<0.001). Representative photographs of PCNA staining of PIL-4 cells grown in control media and media supplemented with 250 IU/mL IFNα are shown in E and F respectively. ......................................................................................................................... 65 Figure 4.3. Changes in cyclin D1 expression in PIL-2 and PIL-4 cells following 24 hour treatment with IFNα. IFNα resulted in a greater than 50% reduction in Cyclin D1 expression in PIL-2 cells (A, **p<0.001). No significant effect of IFNα on the expression of Cyclin D1 by PIL-4 cells was observed (B). Note: mRNA levels of cyclin D1 were normalized against mRNA levels of β-actin. ....................................................................... 66 Figure 4.4. Effect of IFNα on apoptosis of PIL cells. The percentage of apoptotic cells significantly increased in both PIL cell lines following administration of IFNα. A 7-fold increase was seen in PIL-2 cells (A, ***p<0.0001) and 14-fold increase was seen in PIL-4 cells (B, ***p<0.0001)......................................................................................................... 67 Figure 4.5. Effects of IFNα on differentiation of PIL cells. Following 8 days treatment with 250U/mL IFNα, significant changes were detected in the expression of markers specific to various stages of maturation. There was an increase in the expression of the mature markers albumin in PIL-2 cells (A, ***p<0.001) but no significant effect on PIL-4 cells (B). Expression of CK19 increased in both cell lines following IFNα treatment (PIL-2

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C & PIL-4 D, *p<0.05). Note: mRNA levels of hepatic maturation markers were normalized against mRNA levels of β-actin. ....................................................................... 69 Figure 4.6. Treatment of PIL-2 cells with IFNα does not result in morphological changes after 8 days of treatment. Figure 4.6A is a representative photograph of PIL-2 cells after being cultured in medium containing 250 IU/mL IFNα for 6 days at 40X magnification. Figure 4.7B shows PIL-2 cells after being cultured in control Williams E medium for 6 days. Figures 4.6C and D are photographs of PIL-2 cells after 8 days exposure to either IFNα or control medium. ..................................................................................................... 71 Figure 4.7. Treatment of PIL-4 cells with IFNα does not result in morphological changes after 8 days of treatment. Figure 4.7A is a representative photograph of PIL-4 cells after being cultured in media containing 250 IU/mL IFNα for 6 days at 40X magnification. Figure 4.7B shows PIL-4 cells after being cultured in control Williams E media for 6 days. Figures 4.7C and D show PIL-4 cells after 8 days in culture either in the presence or absence of IFNα................................................................................................................... 72 Figure 5.1. Treatment with IFNα reduced numbers of A6-positive cells in mice placed on CDE diet. A6-positive cells decreased by approximately 4-fold in IFNα treated group compared to placebo (*p<0.05, A). Representative photographs taken of A6-positive progenitor cells in placebo and IFNα−treated animals (B & C).......................................... 82 Figure 5.2. Treatment with IFNα reduced numbers of MPK-positive cells in mice placed on CDE diet. MPK-positive cells decreased by approximately 30% in IFNα treated group compared to placebo (*p<0.05, A). Representative photographs taken of MPK-positive HPC cells in placebo and IFNα−treated animals (B & C)................................................... 83 Figure 5.3. Treatment IFNα reduced numbers of c-kit-positive cells in mice placed on CDE diet. The proportion of c-kit-positive cells was reduced by almost 5-fold in the IFNα treated group compared to placebo (**p<0.005, A). Representative photographs taken of c-kit-positive HPC in placebo and IFNα−treated animals (B & C). ............................................ 84 Figure 5.4. Treatment of mice on CDE diet with IFNα reduces proliferative status of progenitor cells while it increases proliferative status of hepatocytes. IFNα treatment reduced the mean percentage of PCNA-positive HPC by 20% (*p<0.05, A), while almost doubling the percentage of PCNA-positive hepatocytes (***p<0.001, B). Representative photographs of PCNA-staining in IFNα (C) and placebo (D) samples. Hepatocytes indicated by red arrows and progenitor cells indicated by black arrows. ............................ 85 Figure 5.5. Treatment with IFNα increases proportion of apoptotic progenitor cells but not hepatocytes. The mean percentage of TUNEL-positive hepatic progenitor cells increased slightly, from 1.1% to 1.6%, however this difference was not significant (A). There was no change in proportion of TUNEL-positive hepatocytes (B). Representative photographs of TUNEL-staining in IFNα (C) and placebo (D) samples. Hepatocytes indicated by red arrows and progenitor cells indicated by black arrows........................................................ 87 Figure 5.6. Treatment with IFNα cause changes in cytokine profile of mice place on CDE diet. Minimal amount of IFNγ is detected following treatment (*p<0.05, A). Levels of LTβ in the IFNα group were less than half of placebo (*p<0.05, B). IFNγ levels were 6 times lower in treated animals compared to placebo (*p<0.05, C). No significant changes in TNFα and IL-6 were observed (D&E). Note: mRNA levels of cytokines were normalized against mRNA levels of β-actin. .......................................................................................... 89

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Figure 5.7. Representative photographs of sirius red staining for fibrosis staging. There was no discernible difference between fibrotic status between IFNα- (A) and placebo-treated mice (B)................................................................................................................................ 90 Figure 5.8. Treatment with IFNα significantly reduced numbers of activated HSC. HSC which stained positive for αSMA were reduced by almost 50% in the IFNα-treated group (*p<0.05, A). Representative photographs of αSMA-positive cells taken from IFNα and placebo animals (B & C)...................................................................................................... 91 Figure 5.9. Treatment with IFNα significantly increased numbers of phospho-STAT-3-positive cells. Cells which stained positive for phospho-STAT-3 were increased by more than 2-fold in the IFNα treated group (***p<0.001, A). Representative photographs of phospho-STAT-3-positive cells taken from IFNα-treated and placebo animals (B & C)... 93 Figure 6.1. Both chains of the IFNγ receptor are expressed by PIL-2 and PIL-4 cell lines. The α chain of the IFN receptor is expressed by PIL-2 and PIL-4 as seen in lanes 1 and 2, respectively. The β chain of the IFN receptor is expressed by PIL-2 and PIL-4 as seen in lanes 3 and 4, respectively. ................................................................................................ 106 Figure 6.2. Interferon γ reduced mitochondrial activity. Following 24 hours culture in media containing a spectrum of concentrations of IFNγ, mitochondrial activity is reduced in both PIL-2 (A) and PIL-4 (B) cell lines. LD50 was 20ng/mL for PIL-2 cells and 5ng/mL for PIL-4 cells. ......................................................................................................................... 107 Figure 7.1. Interferon γ treatment increased numbers of A6-positive cells in mice placed on CDE diet. A6-positive cells increased by approximately 3-fold in IFNγ treated group compared to placebo (**p<0.005, A). Representative photographs taken of A6-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 200X magnification. .... 121 Figure 7.2. Interferon γ treatment increased numbers of MPK-positive cells in mice placed on CDE diet. MPK-positive cells increased by more than 3-fold in IFNγ treated group compared to placebo (**p<0.005, A). Representative photographs taken of MPK-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 400X magnification. .... 122 Figure 7.3. Interferon γ treatment increased numbers of c-kit-positive cells in mice placed on CDE diet. The proportion of c-kit-positive cells increased by more than 3-fold in IFNγ−treated group compared to placebo (***p<0.001, A). Representative photographs taken of c-kit-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 400X magnification............................................................................................................ 123 Figure 7.4. Treatment with IFNγ increases proportion of apoptotic hepatocytes but not hepatic progenitor cells. The proportion of TUNEL-positive HPC was not significantly affected by IFNγ treatment (A). However, it did significantly increase the percentage of TUNEL-positive hepatocytes by approximately 10-fold (B, **p<0.01). Representative photographs of TUNEL-staining in IFNγ (C) and placebo (D) samples at 400X magnification. .................................................................................................................... 125 Figure 7.5. Treatment with IFNγ increased mean numbers of inflammatory cells as shown by CD45 immunohistochemical staining. Treatment of CDE-fed mice with IFNγ increased CD45-positive cells by 4-fold (A, **p<0.01). Representative photographs of CD45 staining in IFNγ-treated (B) and placebo animals (C) at 400X magnification................................ 126 Figure 7.6. Representative photographs of sirius red staining collagen fibres in periportal regions of livers from CDE-fed mice administered 2ng/mL IFNγ daily (A) and vehicle (B) at 200X magnification........................................................................................................ 127

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Figure 7.7. Changes in hepatic cytokine profile following daily administration of 2ng/mL IFNγ to CDE-fed mice. mRNA levels of TNFα were doubled by IFNγ treatment (A), while expression of IL-6 increased by 46-fold (B). Concurrently, IFNγ treatment caused significant decreases in the hepatic expression of LTβ (C), IFNγ (D) and TGFβ (E). Note: mRNA levels of cytokines were normalized against mRNA levels of β-actin.................. 129 Figure 7.8. Mean serum ALT measurements taken from mice administered vehicle (placebo) and 2ng/mL IFNγ daily with CDE or control diet, after 3, 5 and 7 days. Mice placed on control diets did not experience a change in ALT levels even when administered daily intraperitoneal injections of IFNγ. Elevation of ALT levels is expected in the CDE-fed mice, however, administration of IFNγ causes an exaggeration of initial liver damage at day 3, which eventually subsides to basal levels by day 7................................................. 131 Figure 7.9. Treatment with IFNγ induces influx of inflammatory cells. Representative photographs of haematoxylin and eosin-stained tissue sections at 200X magnification. Pockets of inflammatory cells were observed in IFNγ/control animals at Day 3 (A) and to a lesser extent at Day 5 (B). This is no longer observed at Day 7 (C). Normal healthy liver architecture was noted in the placebo/control animals at Day 3, 5 and 7 (D-F respectively)............................................................................................................................................. 133 Figure 7.10 Treatment of CDE-fed mice with IFNγ increased hepatocyte apoptosis. Basal level of hepatocyte turnover is seen in mice on control diets. IFNγ treatment alone did not significantly increase the extent of apoptosis. Mice fed the CDE diet had increased hepatocyte death within the first 5 days of feeding. IFNγ treatment of CDE-fed mice caused an approximate 4-fold increase in the first 3 days compared to those given placebo (*p<0.05)............................................................................................................................ 136 Figure 7.11. Double immunohistochemical staining for Ki67 and cytokeratin identifies the population of actively proliferating HPC population. Treatment with IFNγ significantly increases proliferation of progenitor cells both control- (*p<0.05) and CDE- (**0<0.01) fed mice. ................................................................................................................................... 138 Figure 7.12. Representative photographs (200X maginification) of double immunohistochemical staining with Ki67 and cytokeratin for identification of proliferating hepatic progenitor cells. Cytokeratin-positive cells stain red while Ki67 cells stain blue. Control/Placebo – Day 3 (A), Day 5 (B), Day 7 (C); Control/IFNγ – Day 3 (D), Day 5 (E), Day 7 (F). ........................................................................................................................... 139 Figure 7.13. Representative photographs of double immunohistochemical staining of Ki67 and cytokeratin shown at 400X magnification. Above photographs show double staining of CDE/Placebo (A) and CDE/IFNγ (B) at day 3. Only cells positive for both markers (indicated by black arrows) were included in the cell count illustrated by Figure 7.10. ... 141

xvii

List of Tables Table 1.1. Animal models of oval cell response .................................................................. 17 Table 3.1. Summary of immunohistochemical and cell surface markers expressed by HPC.............................................................................................................................................. 44 Table 3.2. Liver Biopsy Samples From Chronic Hepatitis C Patients................................. 46 Table 4.1. Primers for target gene sequences analyzed by quantitative PCR...................... 63 Table 5.1. Primers for cytokine profile analyzed by quantitative PCR ............................... 79 Table 5.2. Interferon α treatment does not significantly affect the stage of fibrosis in CDE-fed mice................................................................................................................................ 90 Table 7.1. Fibrosis staging of mice placed on CDE diet.................................................... 127 Table 7.2. Additional experimental time points and regimes ........................................... 130

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1

1. General Introduction

1.1. Anatomy, Physiology and Vasculature

The human liver is located in the right upper quadrant, extending from the fifth intercostal

space in the midclavicular line down to the right costal margin. This places it in a unique

position to receive venous blood directly from the intestine, spleen and pancreas where it

encounters toxins, nutrients and hormones. Approximately 70% of the blood flow to the

liver is delivered by the portal vein. There are three main hepatic veins. In most individuals,

the middle and left veins join before entry into the vena cava. In contrast, the major hepatic

artery arises from the celiac artery. Sympathetic and parasympathetic innervation of the

liver are supplied with fibres derived from the lower thoracic ganglia, celiac plexus, vagi

and right phrenic nerve forming the plexuses around the hepatic artery, hepatic portal vein

and bile duct.

The unique sinusoidal structure of the liver is well suited for the bi-directional transfer for a

variety of solutes 1. Absence of basement membranes in the sinusoidal endothelium, as well

as its fenestrae, allow direct contact between portal blood and the Space of Disse.

Interchange of macromolecules between sinusoidal blood and hepatocytes is facilitated by

the microvilli found on the plasma membrane of the sinusoidal endothelium. A key

function of the liver is maintaining glucose homeostasis in the body. It is involved in the

regulation of carbohydrate, protein and fatty acid metabolism and transport.

Chapter 1

2

Figure 1.1. Diagrammatic representation of a liver acinus 2.

The fundamental unit of the liver is the acinus, seen as a central vein surrounded by four to

six portal triads (Figure 1.1). Hepatocytes abutting the hepatic central vein form the

pericentral zone of hepatocytes, while hepatocytes adjacent to the hepatic portal triad are

known as periportal hepatocytes. There are functional differences between pericentral

hepatocytes and periportal hepatocytes in that they exhibit different synthetic capabilities,

expression of biotransformation enzymes and patterns of susceptibility to liver injury.

Diverse hepatocyte function is facilitated by differences in the local environment such as

relative hypoxia and nutrition depletion of sinusoidal blood 3.

Hepatocytes are hexagonal cells with a central nucleus, arranged in plates flanked by

sinusoids. The sinusoidal space is covered by a layer of endothelium which encloses the

extravascular Space of Disse, where lymphocytes and hepatic stellate cells, responsible for

lipid storage, are located. Kupffer cells are hepatic macrophages found in the sinusoids and

Chapter 1

3

have pseudopodia that are anchored onto subendothelial structures. Hepatic sinusoids are

different from systemic capillaries in that they have fenestrated endothelial cells and scanty

subendothelial stroma, which allows for the passage of large macromolecules. The various

cellular components of the liver are discussed in further detail in Section 1.4.

1.2. Liver Development

The development of the liver can first be detected at the 14 to 20 somite stage, when it

appears as an outgrowing bud of proliferating endodermal cells in the ventral floor of the

foregut 4. The bud is separated from the surrounding septum transversum mesenchyme by a

basement membrane 5 which is progressively disrupted as the primary bud develops and

hepatoblasts delaminate from the foregut and migrate as cords into the surrounding septum

travsersum 4,5. As hepatoblasts migrate they closely associate with primitive sinusoidal

endothelial cells that are seen to form capillary-like structures between migrating cords 6.

Electron microscopy of fetal rat liver has shown that cells change from an oblong shape at

day 12-14 of gestation to spherical around day 18 and finally becoming polygonal prior

birth on day 20 7. Differentiation from hepatoblast to hepatocyte is a gradual process and

the change in ultrastructure reflects ongoing differentiation.

Embryological studies have traced the proliferation and differentiation of stem cells into

hepatocytic and biliary lineages during liver development 4,8-11. On embryological day (ED)

8.5 in the mouse, there is proliferation of undifferentiated endodermal cells of the ventral

foregut and migration into the septum transversum where they associate with mesenchymal

cells and form the hepatic diverticulum 12. The foregut-derived cells begin to express α-

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4

fetoprotein (AFP) at ED 9.0 and albumin at ED 9.5 followed by cytokeratins (CK) 8, 14

and 18 8,13. As the cells develop into hepatoblasts they begin to also express γ-glutamyl

transpeptidase (GGT), α1-antitrypsin, π-glutathione-S-transferase (π-GST) and fetal

isoforms of aldolase, lactic dehydrogenase and the M2 isozyme of pyruvate kinase (M2PK)

14-18. The cells proliferate rapidly between ED 12 and ED 16 and subsequently diverge

along either the biliary or hepatocytic lineage, hence this period is referred to as the

differentiation window in hepatic development 19.

1.3. Liver Regeneration

All the cells of the body are not permanent, except terminal cells such as neurons. There is

a constant turnover of cells, with old cells dying and new cells replacing them. Early

estimates showed that there was at least 1 in 20, 000 to 40, 000 cells dividing at anyone

time and that the liver replaced all of its cells in approximately one year 20. Later studies

into liver cell proliferation using 3H-thymidine incorporation showed that proliferation

occurred mainly in the periportal area compared to the areas along the hepatic cord, close to

the central vein. This led researchers to believe that liver renewal involved proliferation of

hepatocytes in the periportal region and streaming of liver cells from the portal to the

central area with terminal differentiation around the central vein 21. Rodent models of liver

regeneration have shown that liver mass lost to surgical resection is regained via the

proliferation of fully differentiated and normally quiescent hepatocytes and bile duct cells

in the unresected tissue 22, a process referred to as compensatory hyperplasia. However,

when hepatotoxic injury impairs the normal replication process, a heterogeneous population

of cells is activated. They are referred to as hepatic progenitor cells (HPC) and are thought

Chapter 1

5

to originate from the Canals of Hering. The resulting network of cells then invade the liver

parenchyma where these cells undergo differentiation forming either hepatocytes or bile

ducts to restore original liver architecture and function 23-30

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6

1.4. Cellular Components of the Liver

The major cellular components of the liver are the hepatocytes and the biliary duct cells.

Approximately 80% of the total cell population in an adult liver are hepatocytes. They

measure 20 – 30µm in diameter with large granular cytoplasm and basophilic nuclei. The

hepatocytes are responsible for most hepatic functions including metabolizing nutrients and

xenobiotics, as well as secreting plasma proteins. Almost 5% of the liver cell population is

comprised of biliary epithelial cells. They form ductular structures of variable sizes, and are

involved in bile production and transport. They are morphologically distinct from

hepatocytes, with a cuboidal shape on cross-section and are of a smaller size compared to

hepatocytes. In addition to these two major cell types, the liver also consists of Kupffer

cells, hepatic stellate cells and hepatic progenitor cells. These cell types will be discussed in

further detail.

1.4.1. Kupffer Cells

Kupffer cells are located in the sinusoidal spaces of the liver and are involved in

immunological and inflammatory responses as well as metabolism of various compounds.

These cells are the resident macrophages of the liver and are the first line of defense against

invading microorganisms. The activation of Kupffer cells is induced by a variety of

different factors including the components released by damaged cells, lipid peroxides from

apoptotic cells, intermediate metabolites of drugs and hepatotoxins, acetaldehyde from

alcohol metabolism as well as reactive oxygen species (ROS). The activated population of

Kupffer cells then expands and releases cytokines to activate hepatic stellate cells (HSC).

Activated Kupffer cells are a major source of two profibrinogenic cytokines – transforming

Chapter 1

7

growth factor β (TGFβ) and platelet derived growth factor (PDGF). They also produce

tumor necrosis factor α (TNFα) that plays a crucial role in mediating inflammation, and to a

lesser extent HSC activation.

1.4.2. Hepatic Stellate Cells

Hepatic stellate cells (HSC) are found in the parasinusoidal space and are responsible for

storing most of the body’s vitamin A 31. HSC are usually quiescent and produce small

quantities of extracellular matrix (ECM) components for basement membrane formation.

They can be activated to become myofibroblast-like cells when exposed to soluble factors

such as TGFβ, PDGF and reactive oxygen species (ROS) produced by infiltrating

neutrophils, damaged hepatocytes and activated Kupffer cells 32-38. The activation of HSC

results in a rapid production of large amounts of ECM components thus triggering the

fibrotic cascade.

HSC play a pivotal role in mediating response to liver injury. Activation, proliferation and

apoptosis of these cells determine the outcome of fibrogenesis. While the embryonic and

extrahepatic origin of HSC remain unknown, this population of cells are currently

suggested to arise from circulating bone marrow cells 39. There has also been suggestion

that HSC play a role in the oval cell response due to the intimate physical interaction

between the two cell types 40-42.

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8

1.4.3. Hepatic Progenitor Cells

Hepatic progenitor cells (HPC) are a spectrum of cells with distinct subpopulations, some

with multilineage potential similar to somatic stem cells and others that have progressed

further down a differentiation pathway and produce progeny of only a single lineage 43.

They are thought to arise from the Canals of Hering where the terminal bile ductules meet

distal hepatocytes. HPC express markers common to biliary ductules such as OV6, CK 7, 8,

14, 18 and 19, π-GST; and hepatocytes such as AFP, albumin, M2PK; and haematopoietic

markers such as stem cell factor receptor (c-kit), CD34 and Thy-1 15,44-50. A summary of

markers expressed by hepatic progenitor cells can be found in Section 3.1. (Table 3.1).

Due to this guilty-by-association relationship, HPC have long been thought to differentiate

into biliary ducts and hepatocytes. While this has been shown to be true 51-53, research has

also shown that this heterogenous compartment of cells is versatile in its differentiation

capabilities. HPC have been shown to undergo small intestinal metaplasia in rats exposed

to 2-AAF 54, as well as undergoing transdifferentiation into pancreatic endocrine hormone

producing cells when cultured in a high glucose environment 55.

In 2001, Sell postulated that there were three levels of proliferating cells in the liver,

namely the mature hepatocytes, the hepatic progenitor cells thought to arise from the

Canals of Hering, and the multipotent stem cells of the liver supposedly derived from

circulating bone marrow cells 56. Stem cells are generally considered to exhibit the

following characteristics: (i) self-renewal, (ii) multipotency, (iii) long term tissue

repopulation following transplantation, and (iv) serial transplantibility 57. Progenitor cells

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9

are the progeny of stem cells. The classical paradigm where intrahepatic cells are

replenished by resident progenitor cells has been recently revised to include extrahepatic

stem cells 58.

Little was known about the liver potential of bone marrow cells until Petersen and

colleagues reported that a stem cell associated with the bone marrow had epithelial cell

lineage capability 59. Shortly after, the same group reported the expression of the

haematopoietic stem cell marker Thy-1 in oval cells 45. In the wake of this groundbreaking

discovery several groups of researchers reported hepatocytes expressing donor markers in

recipient livers following bone marrow transplantation, thus further emphasizing the role of

extrahepatic stem cells in liver repopulation. Briefly, female animals were lethally

irradiated and rescued by bone marrow cells from labeled males or purified haempatoietic

cells. Repopulation of the liver by bone marrow derived cells was thought to occur when Y

chromosome-bearing hepatocytes were detected in the liver 60-64. Similar findings were

reported in humans receiving cross gender transplants 24,65,66. However, later studies using

the Fah -/- mouse model have shown that cell fusion rather than transdifferentiation of bone

marrow derivatives, is responsible for the high levels of liver repopulation 67,68.

Chapter 1

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1.5. Hepatic Progenitor Cell Activation in Chronic Liver

Diseases

Activation of the HPC compartment has been detected in a range of hepatocarcinogenic

liver conditions, such as chronic viral and alcoholic hepatitis 50,69,70, where numbers of HPC

have been shown to correlate to severity of liver disease 69,71. Activation of this

heterogenous cell population only occurs when the regenerative capacity of the liver has

been impaired 23,72-74.

1.5.1. Chronic Hepatitis C

Of the chronic liver diseases that result in a HPC response, chronic hepatitis C is amongst

the most common diseases to be studied. In countries such as the United States and Japan,

chronic hepatitis C infection accounts for at least 50% of all cases of HCC 75,76. More than

80% of all infected individuals develop chronic infection and approximately 20% of all

chronically infected individuals develop cirrhosis in the 10-20 years that follow. Of these

individuals, up to 5% develop HCC each year 76,77.

The hepatitis C virus is an RNA virus belonging to the family Flaviviridae. The genome

consists of approximately 9, 400 nucleotides with one large open-reading frame encoding

for a polypeptide consisting of structural and non-structural domains. A representative

figure of the genomic constitution of the hepatitis C virus is shown in Figure 1.2.

Currently, the gold standard of treatment for chronic hepatitis C is a combination of

pegylated IFNα and ribavirin 78. Generally patients are administered pegylated IFNα

Chapter 1

11

subcutaneously on a weekly basis for 24-48 weeks in combination with ribavirin which is

taken orally.

Several years ago, Tanaka and colleagues reported that IFNα therapy caused a significant

risk reduction of HCC development in chronic hepatitis C patients who achieved

biochemical response 79. In the years following, other researchers demonstrated that IFNα

therapy could reduce HCC development in chronic hepatitis C independent of viral

clearance 80-83. This cytokine has also been shown to decrease oxidative stress in HSC 84,

reduce expression of α1-collagen and collagen accumulation in the rodent model of fibrosis

85 and it has also been recently used to treat fibrosis in gene therapy 86. The signaling

pathways and molecular mechanisms of IFNα are further discussed later in this chapter.

5’ NTR 3’ NTR

Structural proteins Non-structural proteins

C E1 E2

Envelope proteins

Nucleocapsid

NS 1 NS 2 NS3 NS4A

NS4B NS5A NS5B

Metalloprotease Serine protease RNA helicase

Transmembrane protein

Co-factors IFN resistance protein

RNA polymerase

Chapter 1

12

Figure 1.2. A representative diagram of the structure of the hepatitis C virus genome. The genome carries an open reading frame (ORF) encoding a polypeptide precursor of 3010 amino acids. Its translation is directed via a ~340 bp nucleotide long 5’ non-translated region (NTR), which functions as an internal ribosome entry site. It allows for direct binding of ribosomes in close proximity to the start codon of the ORF. The polypeptide is then cleaved to form 10 products. The structural proteins, C, E1 and E2 at the first third from the N-terminal, and the non-structural replicative proteins, N2-5, located further. Putative functions of these proteins are stated above.

Chapter 1

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1.5.2. Hepatic Progenitor Cells as Targets of Malignant Transformation

Coined “oval cells” in rodents, HPC were first described in 1956 by Farber and colleagues

when they found oval-shaped cells appearing in the livers of rats subjected to carcinogenic

diets. The same research group later developed a model of cancer induction in the rat liver

where rapid expansion of initiated cells into hepatocyte nodules was achieved using the

selective inhibition of surrounding tissue 87. Rats which had been treated with carcinogens

produced a rare population of HPC when briefly exposed to an agent that blocked normal

hepatocyte cell division, such as 2-AAF. The resulting oval cells were able to withstand

growth inhibitory effects of chemicals such as 2-AAF and proliferate upon stimulation 87,88.

This established a new principle in the dedifferentiation theory of cancer development, that

is, differential growth inhibition of surrounding cells could form the basis of the emergence

of focal proliferative lesions 87, a precursor of neoplasia. This was thought to require three

components: (i) the induction of oval cells with resistance towards cytotoxicity; (ii)

imposition of a growth constrained environment for most cells in the surrounding tissue and

(iii) a strong selective pressure in the form of a growth stimulus 87.

Hepatocellular carcinoma (HCC) has been extensively studied over the past several

decades. The cellular origin of HCC is still an open question. There are generally two

schools of thought regarding the cellular origin of liver cancer, given the fundamental

principle that cancer must arise from cells that have retained their proliferative capacity,

namely the dedifferentiation of terminally differentiated cells or the maturation arrest of

transit-amplifying cells from a malignant stem cell.

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14

Detailed immunophenotyping studies have demonstrated that a substantial proportion of

human HCC express progenitor cell markers 70,89-92. This could be attributed to an

acquisition of progenitor cell markers during malignant transformation by mature

hepatocytes (dedifferentiation hypothesis) or progenitor cells differentiating down the

hepatocytic lineage (maturation arrest hypothesis). Ease of transformation of these cells in

culture has been previously documented 93,94 and a number of HCCs express HPC markers

95-98 suggesting that this compartment of cells are targets to malignant transformation in

instances of chronic liver injury.

Experimental hepatocarcinogenesis has been used to show the sequence of neoplastic

changes; from foci to nodules and finally to cancer, as well the associated changes in

enzymatic content of preneoplastic and neoplastic cells 99-108. The maturation arrest model

arises from the theory that cancer is a result of blocked ontogeny; the failure of cells to

differentiate. Morphologically, the neoplastic focus retains the morphology of the cell

lineage from which it originated and reflects the stage of maturation at which ontogeny was

blocked. The concept of maturation arrest was developed in order to explain the similarity

in appearance between cancers and embryonic tissues. The supporting evidence for this can

be found in rare teratocarcinomas which arise from multipotent stem cells which have the

capacity to differentiate into non-malignant cells representing almost every adult tissue in

the body 109-111.

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Activation of the HPC compartment preceeds HCC development in animals placed on

carcinogenic regimes. The neoplastic foci in these animal models express oval cell markers

including OV-6 and AFP 26,56,98,112,113, suggesting that oval cells are a potential target for

hepatocarcinogens. Isolation, transformation and transplantation of oval cells have shown

that they can result in HCC. Additionally, c-myc, c-Ha-ras, p53 and connexins appear to

play vital roles in the malignant transformation of oval cells 49,93,94,114. Activation of HPC

has also been observed in pro-carcinogenic conditions including chronic viral hepatitis and

alcoholic liver disease, with increased oval cell numbers corresponding to severity of liver

disease 50,69,70. The precursor-product relationship between HPC and HCC has been further

confirmed by a study conducted by Shachaf and colleagues where inactivation of oncogene,

c-myc, resulted in en masse differentiation of tumor cells into biliary ducts and hepatocytes,

accompanied by the loss of AFP expression 115.

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1.6. Models of Progenitor Cell Activation

The type of liver injury appears to play a major role in determining whether progenitor cells

differentiate into hepatocytes (plasticity), fuse with other hepatocytes (fusion) or transform

into other cell lineages (trans-differentiation) 116,117. Table 1.1 summarizes the various

animal models of oval cell activation in rodents. They include a variety of chemical

regimes such as administration of dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-

AAF) by oral gavage, feeding of a choline-deficient (CD) or choline-deficient ethionine

supplemented (CDE) diet, 3’-methyl diaminobenzidine (3’-Me-DAB) diet or even a

combination of two or more chemical carcinogens and hepatotoxic agents. The extent of

liver injury and thus HPC response is often exacerbated by physical challenge such as 2/3

partial hepatectomy (PH). Some researchers prefer using methods of injection of the

carcinogens such as the regimes which involve intraperitoneal (i.p.) injections of

galactosamine, dipin and diethylnitrosamine (DEN). Less frequently, and perhaps due to

issues of cost, there are those who use transgenic mice for models of HPC cell response.

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Table 1.1. Animal models of oval cell response

REGIME OBSERVATION SOURCE 2-AAF Male and female rats were given 10mg/kg 2-AF, 2-AAF or N-OH-AAF by oral gavage for 1-9 days

Oval cells proliferated within 24 hours of exposure to carcinogen. No expansion and migration of these cells were observed.

118

2-AAF/CCl4 Male rats were fed a diet supplemented with 0.02% 2-AAF. CCl4 (2mL/kg) given by oral gavage on day 7 and killed 1-14 days after CCl4

Massive oval cell proliferation from periportal regions accompanied parenchymal necrosis. Oval cells disappeared following withdrawal of carcinogen.

119

2-AAF/CD diet Male rats were fed a CD diet supplemented with 0.05% 2-AAF for 12 days, then returned to a normal diet and sacrificed after 28 days after commencing regime

Proliferation of oval cells with no complete hepatocyte differentiation observed. Results were very similar to those seen in 2-AAF/PH models.

120-123

2-AAF/PH Male rats were fed 2-AAF (average daily dose 6.6mg/kg) for 5 days, PH and feeding continued for following 4 days. Animals were sacrificed 13 days following PH.

Oval cells numbers gradually increased following PH. No parenchymal necrosis was detected. Labeled oval cells differentiated into basophilic hepatocytes; establishing the precursor-product relationship between oval cells and hepatocytes. Oval cells expressing AFP underwent intestinal metaplasia. AFP expression was not seen in metaplastic tissue.

124 125

Male rats were fed a normal diet supplemented with 0.02% 2-AAF for 14 days and PH administered midway. Animals were sacrificed 9 weeks following PH.

Oval cells proliferated into parenchyma with intestinal metaplasia often observed after 3 weeks. Metaplastic tissue developed into cholangiofibrotic lesions.

54

Male rats were fed 2-AAF by oral gavage (10mg/kg) for 14 days with PH performed midway. Animals were sacrificed 7 days following PH.

Massive oval cell proliferation and intestinal metaplasia was seen 1 week following PH. Hepatocyte differentiation was seen in some animals

126,127

Male rats were fed 2-AAF (either 2.5mg/kg or 5mg/kg) from 6 days prior to and up to 7 days following PH. Animals were sacrificed 14 days following PH.

5mg/kg 2-AAF diet caused a similar response as did 10mg/kg; described above. In animals fed 2.5mg/kg 2-AAF, intestinal metaplasia was absent while most oval cells differentiated into small hepatocytes by day 14.

23,128

0.1% ethionine - resulted in proliferation of oval cells but did not form clear ductular profiles. Differentiation into hepatocytes was rare and no accompanying parenchymal necrosis was detected.

129,130

0.07% ethionine - caused proliferation of oval cells which expressed both adult and fetal markers. The oval cells first appeared as cords, then developing into ducts by 2 weeks and intestinal-like cells by 5 weeks.

131

CDE diet Male rats were fed a CD diet supplemented with 0.05-0.1% ethionine for 1 day to 12 weeks

0.05% or 0.1% ethionine – massive oval cell proliferation accompanied by cholangiofibrosis and parenchymal necrosis. Earlier onset and more extensive damage seen in higher dose.

132

Massive proliferation of oval cells penetrated the parenchyma with no accompanying necrosis. Intestinal metaplasia and cholangiofibrosis was observed after 12 weeks

133

3’-Me-DAB Male rats were fed a normal diet supplemented with 0.06% 3’Me-DAB for 12 weeks.

Proliferation of oval cells accompanied by hyperplasia of small hepatocytes, which expressed GGT possibly derived from oval cells. By week 7 of feeding numbers of oval cells decreased and were replaced by GGT-expressing hyperplastic hepatocyte foci.

134

Galactosamine Male rats were given a single i.p injection of galactosamine (700mg/kg). Animals were sacrificed 1-8 days following dose.

Proliferation of oval cells accompanied by parenchymal necrosis. Oval cells lose their biliary specific markers and differentiate into transitional hepatocytes.

135,136

Male rats were given two i.p. injections of galactosamine (750mg/kg) 6 hours apart. Animals were sacrificed 1-10 days later

Similar observation as described above. 137

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Table 1.1 Animal models of oval cell response (continued)

REGIME OBSERVATION SOURCE Dipin Male mice were given a single i.p. injection of dipin (60mg/kg) and PH 2 hours later. Animals were sacrificed 18 months thereafter.

Oval cell proliferation accompanied by initial parenchymal necrosis. Small hepatocytes are derived from oval cells and eventually replace damaged parenchyma.

138

CCl4 inhalation No further details given on dosage.

Centrilobular necrosis was observed with oval cells appearing as small ducts and clusters of cells in the perinecrotic areas.

139

DEN Male mice of three strains were given a single i.p. injection of DEN (10-150mg/kg). Animals were sacrificed 1-7 days following dose.

Magnitude of hepatic necrosis and oval cell response varied between strains. Differentiation of oval cells into hepatocytes was observed.

140

Long term exposure to ethanol Rats were fed on a liquid diet supplemented with 5% ethanol for 1-24 months

Oval cells were sparse and proliferated very slowly. They expressed fetal and mature hepatocyte markers. Changes were similar to those seen in CDE diet by over a much longer period

141

Allyl alcohol Female rats were given a single i.p. injection of allyl alcohol (0.62mmol/kg). Animals were sacrificed 6 hours to 6 days following dose.

Periportal necrosis was detected within 6 hours of injury. Small hepatocytes derived from oval cells replaced damaged parenchyma.

142

DMN and Clonorchis sinesis Syrian hamsters were infected with metacercariae of C. sinesis and given DMN (15ppm) in drinking water for 28 days

Intense proliferation of oval cells accompanied by ductular dysplasia and development of cholangiocarcinoma.

143

P21CIP1/WAF1 transgenic mouse P21 overexpression targeted to the liver

Oval cell response with retarded liver development. 144

Long-Evans Cinnamon rat Defect in Wilson disease gene resulting in toxic accumulation of copper. Spontaneous hepatitis occurs as a result.

Acute hepatitis and death. Oval cell proliferation seen in survivors with chronic hepatitis.

145,146

Adenoviral vector/Ganciclovir Male rats were given an intravenous injection of adenoviral vector expressing HSV-tk (4X104 pfu/kg) and ganciclovir given via i.p. osmotic pump (30mg/kg/day). Animals were sacrificed between 1-36 weeks.

Severe hepatocellular damage detected by week 2 with gradual proliferation of oval cells peaking at week 5. Rats with moderately elevated serum transaminases recovered normal liver architecture within weeks, while rats with severe liver damage exhibited persistent oval cell proliferation and eventual cholangiofibrosis and cholangiocarcinoma.

41

Chapter 1

19

1.7. Hepatic Progenitor Cell Lines

A number of HPC cell lines are currently available. The first line of HPC to be established

was a diploid epithelial cell line called WB-344 developed by Tsao and colleagues in 1984.

This cell line was developed from oval cells isolated from an adult male Fisher 344 rat.

Ultrastructurally, they appeared as polygonal cells and expressed aldolase A and C, and

M2-PK 147.

Strick-Marchand and colleagues developed an immortalized, non-transformed bipotential

cell line of HPC isolated from mice known as bipotential murine embryonic liver (BMEL)

cells, which express oval cell markers such as Thy-1, c-kit, AFP and CD34 148. Their ability

to contribute towards liver regeneration and differentiate as bile ducts and hepatocytes has

also been demonstrated 51. In contrast, Dumble and colleagues developed several

transformed progenitor cell lines from oval cells isolated in p53-null mice placed on the

CDE diet. These cells were thus named p53-null immortalized liver (PIL) cells 93. The PIL-

1 and PIL-2 cell lines were obtained from a centrifugal elutriation fraction that contained

pure oval cells while PIL-3, PIL-4 and PIL-5 were derived from fractions containing

clusters of oval cells and hepatocytes. In terms of their tumorigenicity, subcutaneous

injections of PIL-1 and PIL-5 resulted in tumor formation within 2 weeks while requiring 7

weeks in the case of PIL-2. The cell lines designated PIL-3 and PIL-4 were unable to

produce tumors within 3 months of inoculation with 8 X 106 cells 93. A wild-type version of

PIL cell lines has recently been established by Tirintiz-Parker and colleagues (manuscript

in preparation). More recently, Yasui and colleagues developed a rat progenitor cell line

designated C15-5 using Long-Evans Cinnamon rats carrying a defect in the Wilson disease

Chapter 1

20

gene, resulting in hepatic accumulation of copper resulting in acute hepatitis at

approximately 4 months of age. Survivors develop chronic hepatitis accompanied with

massive oval cell response 145.

The first human bipotential liver progenitor cell line known as HepaRG was established

from a liver tumor associated with chronic hepatitis C by Parent and colleagues 149. This

progenitor cell line expressed markers which correlated closely with its murine counterparts

including CK18, CK19, M2PK, OV-1, OV-6 and CD34. More recently a human fetal

progenitor cell line has been established (Yeoh, personal communcation)

Hepatoblast cell lines have also been established - the cell line HBC-3 was developed from

hepatic diverticuli by Rogler after dissecting liver diverticuli from large numbers of E9.5

C57Bl6/DBA embryos and plating onto fibroblast feeders in Dubelcco’s modified Eagle’s

medium (DMEM) supplemented with 10% fetal calf serum (FCS) and β-mercaptoethanol

150. These cells also produced albumin, AFP, a panel of cytokeratins and γ-glutamyl

transferase (GGT). Recently, a human fetal liver multipotent progenitor cell (hFLMPC)

population was isolated and characterized by Dan and colleagues 151. This cell line was

developed from human fetal livers (74-108 days of gestation) dissociated and maintained in

culture where the cell line was kept in an undifferentiated state by means of a feeder layer.

The hFLMPC were demonstrated to differentiate into liver and mesenchymal cell lineages.

Transplantation studies also showed that they were able to differentiate into functional

hepatocytes in vivo.

Chapter 1

21

1.8. Inflammatory Cytokines and the Progenitor Cell Response

Cytokines, or growth factors are specific products of the immune system. They are

polypeptides that act in three short range modes, namely, paracrine, autocrine and

juxtacrine and are hence distinct from classical endocrine hormones 152. Cytokines

modulate cell growth, differentiation and immune defenses of vertebrates. The evolution of

cirrhosis from chronic inflammation has been described as a representation of a dysmorphic

response to injury and it is therefore necessary to understand how inflammatory cytokines

are associated with such a response. In recent years, the close relationship between

inflammatory cytokines and the HPC response has become more firmly established

42,50,69,153. A recent study of cytokine expression during the HPC response revealed that the

expression of TNFα, IL-6, Oncostatin M (OSM), IFNγ and LTβ increased in direct

correlation to the expansion of the progenitor cell compartment during the first 2 weeks of

CDE feeding 154-158. Knockout mice have been employed to definitively test the

requirements of these cytokines for the HPC response. The results show that there is a

critical requirement for TNFα and LTβ, as well as IL-6 and IFNγ to a lesser degree

155,157,159. The first three cytokines will be discussed below, while IFNγ will be discussed in

Section 1.10.

Chapter 1

22

1.8.1. Tumor Necrosis Factor-α

TNFα expression is upregulated during the expansion of the progenitor cell compartment

and is reduced when the cells begin to differentiate 154. This could be due to its differential

effect on progenitor cells at various stages of maturation. It has a pro-proliferative effect on

the immature, undifferentiated progenitor cell, but exerts a pro-apoptotic effect on the cells

as they commit to hepatic differentiation 160. Its role as an upstream inducer of IL-6

signaling in acute liver regeneration has been previously reported 161. TNFα signaling via

the TNF receptor 1 has been shown to be essential to the hepatic progenitor cell response

155. It has also been reported that TNFα induces DNA replication in growth-arrested HPC

through activation of NF-κB and STAT-3 and an increase in expression of c-myc and IL-6

162.

1.8.2. Lymphotoxin-β

LTβ is a major player in inflammation and its promoter contains a functional NF-κB motif

thus rendering it a likely target of pro-inflammatory cytokines 163. LTβ is not generally

detected in the adult liver, although it is seen in the murine fetal liver 164. Elevated levels of

LTβ has also been detected in HPC and small hepatocytes in livers of chronic hepatitis C

patients 156. A recent study by Subrata and colleagues (2005) showed that LTβ is expressed

by both HPC and HCC cells, and basal expression of LTβ is inducible by IL-6 and IL-1β

via key cis-acting promoter elements and regulatory transcription factors. Additionally,

LTβ has been suggested to play a key role in the progenitor cell response 157.

Chapter 1

23

1.8.3. Interleukin-6

IL-6 has been established as one of the cytokines which play a critical role in the HPC

response. Defects in mitosis of hepatocytes in IL-6 -/- mice have been linked to

abnormalities in the G1 phase, absence of STAT-3 activation as well as reduced expression

of c-myc and cyclin D1 165,166. Using dexamethasone, a potent inhibitor of IL-6 and TNFα,

Nagy and colleagues showed that the HPC response is greatly diminished when the activity

of the cytokines is impaired 167. Additionally, IL-6 has been shown to have a pro-

proliferative effect on progenitor cells and it has been suggested to play a role in sustaining

the HPC response via an autocrine mechanism 154,168. In contrast a greater degree of

proliferation has been observed in the HPC compartment of IL-6 -/- mice 169 thus

emphasizing the complexity of this putative stem cell response.

1.8.4. Oncostatin M

OSM is a member of the IL-6 family. The murine receptor of OSM comprises of a gp130

subunit, a commonality of all members of the IL-6 family as has been extensively reviewed

170-172. It is expressed by Kupffer cells and HPC although its effect on progenitor cell

differentiation is currently a subject of debate. While there has been evidence that OSM

stimulates differentiation of rodent and human hepatoblasts, as well as rodent HPC, into

mature hepatocytes 173-176, it appears that it does not exert the same effect on all rodent

models of HPC activation158.

Chapter 1

24

1.9. Interferon α

The interferons (IFN) are a family of proteins that regulate innate immunity, immune

surveillance and homeostasis of peripheral blood populations. There are at least five classes

− IFNα, IFNβ, IFNγ, IFNτ and IFNω. These are divided into two groups; Type I and Type

II. Type II only consist of IFNγ, while all others belong to Type I. IFNα was the first

cytokine to be synthesized using recombinant DNA technology and has been widely used

in the treatment of several neoplasms including renal cell carcinoma and multiple myeloma

177-180. It has emerged as an important regulator of proliferation and apoptosis of liver

cancer cells, affecting cellular communication and signal transduction pathways 181-184.

1.9.1. Interferon Signaling Pathways

Type I (α and β) and Type II (γ) IFN signal through related but distinct pathways.

Type I interferon signaling relies on both receptor chains, denoted IFNαR1 and IFNαR2.

The proteins Janus kinase 1 (Jak1), signal transducers and activators of transcription 1 and

2 (STAT-1 and STAT-2) weakly interact with the intracellular domain of IFNαR2. Binding

of IFNα to its receptor induces the phosphorylation of tyrosine kinase 2 (Tyk2), which is

preassociated with the cytoplasmic tail of IFNαR1, and Jak1 185,186. Activated Tyk2 then

phosphorylates the tyrosine residue at position 466 of IFNαR1, which serves as an anchor

for STAT-2 187. Tyrosine phosphorylation of STAT-2 creates a binding site for STAT-1

which is then transferred from IFNαR2 to STAT-2. Jak1 then phosphorylates STAT-1 188.

STAT-1/STAT-2 complexes associate with a p48 protein to form the interferon-stimulated

gene factor 3 (ISGF-3) which induces transcription when it recognizes an interferon

Chapter 1

25

stimulated response element (ISRE) in the promoter region of an interferon responsive gene

189.

Figure 1.3. Schematic diagram showing the molecular pathways used for interferon α signaling 190.

The effects of IFNα also involve the use of the stress kinase cascade. In addition to the

STAT pathway, Type I IFN can activate members of the mitogen-activated protein kinase

(MAPK), which include Erk and p38 MAPK 191-194. In primary human haematopoietic

cells, IFNα rapidly activated p38 and its downstream effector thus exerting its suppressive

effect on normal haematopoiesis. When the activation of p38 MAPK is inhibited, type I

IFN-dependent inhibition of haematopoietic progenitor colony formation is reversed 195,196.

Chapter 1

26

1.9.2. Effects on Tumor Cell Lines

In addition to its antiviral effects IFNα, has been shown to inhibit growth and induce

apoptosis in several human liver cancer cell lines. Yano and colleagues used a total of 11

human HCC and 2 mixed HCC and CCC cell lines to establish the growth suppressive

effects of IFNα on malignant cell types as well as their expression of the type I IFN

receptor 183. They found that the degree of growth suppression was proportional to the

dosage of IFNα, and 4 basic patterns of growth suppression was observed: (i) blockage of

cell cycle at S phase in 2 cell lines; (ii) blockage of cell cycle at S phase and induction of

apoptosis in 9 cell lines, (iii) blockage of cell cycle at G2/M phase in 1 cell line; and (iv)

blockage at G1 phase. Similarly, Murphy and colleagues found that IFNα inhibited

proliferation of HCC cell lines in a time- and dose-dependent manner by delaying S-phase

progression. Their research suggested that this was most likely to be mediated through

inhibition of specific cyclin-dependent kinases such as cyclin A, and reduced activity of its

associated Cdk2 and Cdc2 kinases 181. IFNα-induced apoptosis was later shown to be

strongly associated with the activation of caspases-1, -2, -3, -8 and -9 accompanied by loss

of mitochondrial membrane potential with release of cytochrome c. Apoptosis of these

malignant cells was also determined to occur independently of interactions between the Fas

receptor and Fas ligand 197.

Chapter 1

27

1.10. Interferon γ

In both humans and mice, a single copy of the IFNγ gene generates a 1.2kb mRNA strand

encoding a polypeptide of 166 residues including a cleaved hydrophobic signal sequence of

23 residues 198,199. IFNγ is responsible for a variety of cellular activities such as the

regulation of several immune responses including the bactericidal activity of phagocytes

and antigen presentation, leukocyte trafficking and effects on proliferation and apoptosis

200,201.

Functionally active IFNγ is a homodimer that binds to two IFNγ receptor 1 (IFNγR1)

subunits, generating binding sites for two IFNγR2 subunits 202,203. The intracellular domains

of the receptor subunits are brought within close proximity to the inactive Jaks they carry,

within the resulting ligand-receptor complex. The Jaks are then sequentially activated by

sequential auto- and trans-phosphorylation, with the activation of Jak2 occurring first as it

is necessary for the subsequent activation of Jak1 204. Once activated, the Jaks

phosphorylate a functionally critical, tyrosine-containing five-residue sequence near the C

terminus of IFNγR1, hence forming paired ligand-induced docking sites for STAT-1. Two

STAT-1 proteins then bind onto these sites because the SH2 domain of each recognizes the

tyrosine phosphorylated sequence 205-207. Receptor-associated STAT-1 is phosphorylated by

the receptor-bound kinases at tyrosine 701, near the C terminus 205,208,209. Activated STAT-

1 homodimers bind to gamma-activated sequences (GAS) elements of IFNγ inducible genes

and stimulate their transcription 210,211. A schematic diagram of this Jak-STAT signaling

pathway is provided in Figure 1.4.

Chapter 1

28

Figure 1.4. Schematic diagram of IFNγ signaling via the JAK-STAT pathway 203.

Chapter 1

29

1.10.1. Interferon γ in the Treatment of Fibrosis

Liver fibrosis is the final common pathway for a large number of chronic liver diseases and

the most effective method of treatment is generally accepted to be the removal of stimulus.

Fibrosis is a wound healing process characterized by the excessive deposition of

extracellular matrix (ECM) proteins generally classified into 3 families – proteoglycans,

glycoproteins, and collagens 212,213. HSC also play a vital role in fibrogenesis, being the key

producers of collagen upon activation during the inflammatory process 214-217.

IFNγ has been used to treat fibrosis by reducing activation of HSC and subsequent collagen

production in experimental hepatic fibrosis as well as in chronic hepatitis B viral infection

218,219. It was in 1987 that exogenous IFNγ was first recommended as a method of treatment

for fibrosis 220. In the years that followed, cytokines received increased interest as possible

treatments for fibrosis and became more widely recognized as a possible tool in the fight

against what has been considered a challenging liver injury process. It has since been

shown that IFNγ reduces fibrosis through any one of a combination of the following: (i)

antagonizing the effect of pro-fibrogenic cytokines and growth factors such as TGFβ, (ii)

inhibiting the synthesis and accumulation of collagen and (iii) interfering with the

activation of HSC 218,219,221,222.

Chapter 1

30

1.10.2. Recent Interest in Role of Interferon γ in Hepatic Progenitor Cell

Response

IFNγ has been shown to be essential for an optimal HPC response. The network of genes

controlled by IFNγ involved in liver regeneration from HPC was first identified by

Bisgaard and colleagues, where they determined that several genes are modulated when

these cells are activated during chronic liver injury 223. These genes included: (i) IFNγ

receptor subunits, (ii) primary and secondary response genes which include gp91phox, IL-

1β converting enzyme (ICE), intercellular adhesion moledcule-1 (ICAM-1) and urokinase-

type plasminogen activator receptor (uPAR), (iii) cytokines which induce the expression of

IFNγ including IL-1β and IL-18, and (iv) cell adhesion molecules responsible for

lymphocyte-epithelial cell interactions including lymphocyte function-associated molecule-

1α (LFA-1).

The expression of IFNγ correlates strongly to progenitor cell numbers in a mouse model of

chronic liver injury where it was shown to play a vital role HPC-mediated liver

regeneration but not in hepatocyte-mediated regeneration 157. IFNγ was also shown to exert

a differential effect on the regulation of cell proliferation of hepatocytes and HPC - two cell

types which rarely proliferate concurrently despite having similar responses to growth

factors 224. In this way, IFNγ has been suggested to be one of the key factors that

determines the type of regenerative response during instances of chronic liver injury.

Levels of IFNγ may determine whether hepatocyte- or progenitor cell-mediated

regeneration occurs to replace loss of functional tissue.

Chapter 1

31

1.11. Aims of Project

The general aims of this project were to determine the effects of IFNα and IFNγ on HPC in

vitro and in vivo. These two interferons are of great interest for two very different reasons.

Pegylated IFNα, in combination with ribarvirin, is the gold standard of treatment for

chronic hepatitis C infection, a common cause of HCC. In an increasing number of case

studies, IFNα has been shown to reduce the risk of HCC development regardless of a

patient’s virological response. Since HPC have long been hailed the cellular targets of

malignant transformation in liver cancer, it was logical to explore the effect of IFNα on this

population of cells.

On the other hand, IFNγ is a cytokine that has generated recent interest in the area of HPC

research. It plays a critical role in the progenitor cell response and has been suggested to be

a key factor that determines the type of regenerative response (hepatocyte- or progenitor

cell-mediated) in the event of liver injury. IFNγ has also been used for treatment of fibrosis,

an event that commonly coincides with HPC response. Together, these findings provided

the premise for an in-depth study into the effects that IFNγ exerts of the HPC population.

Chapter 2

32

2. General Materials and Methods

2.1. Immunohistochemical Methods

2.1.1. Tissue Fixation, Sectioning and Deparaffinization

Generally, tissues were either fixed in 10% phosphate buffered formalin for 12 to 18

hours or Carnoy’s fixative (1 part glacial acetic acid with 3 part absolute ethanol) for 2

hours. Tissues were then further fixed, cleared and impregnated with paraffin using

Tissue-Tek VIP-E150 (Sakura Finetek, Tokyo, Japan). Tissue sections were cut to a

thickness of 5 µm and deparaffinized using 3 changes of xylene and 2 changes of

absolute ethanol, followed by 2 changes of 70% before rehydrating the sections in water.

For frozen sections, tissues were embedded in cryomatrix (Tissue-Tek OCT, Sakura

Finetek, Japan) and snap-frozen in liquid nitrogen.

2.1.2. Antigen Retrieval

Antigen retrieval was performed in one of three ways – tissue sections were either boiled

in EDTA buffer (1mM EDTA, pH 8.0) or citrate buffer (10mM citric acid, pH 6.8) for 15

minutes as previously described 225. Alternatively, tissue sections were exposed to

20µg/mL proteinase K solution in a humidified chamber for 20 minutes at 37oC.

Chapter 2

33

2.1.3. Tissue Section Mounting and Microscopy

Tissue sections were either dehydrated and cleared through 2 changes of 70% ethanol, 2

changes of absolute ethanol and 3 changes of xylene before mounting in DPX mountant

(Sigma-Aldrich, St Louis, MO), or mounted in Kaiser’s glycerol. Microscopy was

performed using a Nikon TE2000-U (Nikon, Kawasaki, Japan). Three non-overlapping

fields were counted at 400X magnification (Field diameter 0.5mm). Cells were scored

when they satisfied the morphological criteria for HPC and were immunoreactive for c-

kit, π-GST or CK19 but did not stain for LCA. Photographs were taken using a Nikon

DS-5M-LI (Nikon).

2.2. Cell Culture Techniques

2.2.1. Maintenance of Cell Cultures

p53-immortalized liver cells lines 2 and 4 (PIL-2 &-4) were cultured in Williams E media

(Sigma-Aldrich), supplemented with ITS+TM Premix (Becton Dickinson, Rockville, MD,

USA), 1%(v/v) Penicillin-Steptomycin (Invitrogen, Auckland, NZ), Fungizone

(Invitrogen), Epidermal Growth Factor (1µg/mL), dexamethasone (10-7M) (Sigma-

Aldrich) and 10%(v/v) fetal bovine serum (Invitrogen). Cells were incubated overnight at

37oC to allow for adherence. Exhausted cell culture media was replaced with serum-free

supplemented Williams E media following serum starvation as described in the following

section.

Chapter 2

34

2.2.2. Serum Starvation

PIL cells were washed in sterile acid wash buffer (50mM glycine, 150mM NaCl and

1mg/mL polyvinylpyrrolidone), re-fed with serum-free Williams E media and incubated

overnight.

2.3. Molecular Techniques

2.3.1. RNA Isolation from Cells

The UltraspecTM RNA Isolation System was used to extract RNA from cultured cells,

according to manufacturer’s instructions (Biotecx Laboratories Inc., Houston, TX, USA).

Briefly, cells were grown on 6-well cell culture plates. The cells were homogenized by

adding 1mL of UltraspecTM to each well and passing cell lysate through a pipette tip

several times. Homogenized cells were transferred into 1.5mL microfuge tubes and

incubated on ice for at least 5 mins. Next, 200µL of chloroform was added to each tube

and the mixture shaken vigorously before incubation on ice for at least 5 mins. The

homogenate was centrifuged at 12, 000g for at least 20 mins. The clear upper aqueous

phase was transferred into a clean microfuge tube. To each tube, an equal volume of

isopropanol was added, mixed by inversion and incubated on ice for at least 15 mins. The

samples were centrifuged again at 12, 000g for 10 mins. The supernatant was discarded

and the pellet was rinsed in 75% ethanol in RNase-free water. The pellet was left to dry

and later resuspended in 50µL of RNase-free water.

Chapter 2

35

The same procedure was used to isolate RNA from tissues, except tissues were first

homogenized in UltraspecTM.

2.3.2. RNA Isolation From Liver Tissue

Approximately 1mg of frozen mouse liver was homogenized in 500µL chilled RNAwiz

(Ambion, Austin, TX) using a glass homogenizer. The mixture was transferred into a

microcentrifuge tube, incubated at room temperature for 15 mins and centrifuged at 14,

000 xg for 20 mins at 4oC. The upper aqueous phase containing isolated RNA was

transferred into a clean tube and an equal volume of nuclease free water was added.

Twice the volume of isopropanol was added to precipitate nucleic acids. This was

incubated at 4oC for a minimum of 10 mins and then centrifuged at 14, 000 x g for 20

mins at 4oC to pellet precipitated RNA. The supernatant was removed and the pellet was

washed with 70% ethanol. Finally, all supernatant was removed and the RNA pellet was

air dried and resuspended in 20µL nuclease-free water and incubated at 60oC for 15 mins

before storing at –20oC until further use.

Chapter 2

36

2.3.3. DNAse Treatment of RNA

Genomic DNA was removed from isolated RNA using DNA-free TM (Ambion). Each

10µL reaction contained 1X Dnase I Buffer and 2 units of rDNaseI. The reactions were

incubated at 37oC for 30 minutes before adding 2µL of DNAse Inactivation Reagent.

This was then incubated for 2 minutes at room temperature with occasional mixing

before it was centrifuged at 10, 000 x g for 90 seconds and the supernatant transferred

into a new tube.

2.3.4. cDNA Synthesis

cDNA was synthesized using ThermoScriptTM RT-PCR System (Invitrogen, Auckland,

NZ). Briefly, each 20uL cDNA synthesis reaction mix included at least 1µg of RNA, 50

µM Olido(dT)20 primer, 1mM dNTPs, 1X cDNA synthesis buffer, 50mM DTT, 40U

RNaseOUTTM and 15 U ThermoScriptTM. Reactions were incubated for 60 minutes at

50oC and terminated by incubating at 85oC. Synthesized cDNA were used for

quantitative PCR immediately.

Chapter 2

37

2.3.5. Cloning Gene Products

It was necessary to clone the PCR products to make plasmid standards in order to

quantify mRNA levels using quantitative PCR. The desired gene product were cloned

into plasmids using the pGEM®T Easy Vector System (Promega, Madison, WI). Briefly,

purified PCR products were ligated with 50ng pGEM®T Easy Vector in a 10uL reaction

mix containing 1X ligation buffer, 3 Weiss units T4 DNA ligase, 3µL purified PCR

product and incubated overnight at 4oC. The resulting reaction mix was diluted 1:5 and

added to 50µL competent DH5α cells and electroporation was performed at 2450V for 5

milliseconds. The cells were then quickly transferred into 1mL SOC broth and incubated

at 37oC in a shaking incubator for an hour. Cells were then plated out on LB plates

containing 0.5mM IPTG, 80µg/mL X-Gal, 100µg/mL ampicillin. Plates were incubated

overnight at 37oC and white transformed colonies were picked and inoculated in 6mL LB

broth supplemented with 100µg/mL ampicillin, and incubated overnight. Plasmids were

then screened as described in the following section.

Chapter 2

38

2.3.6. Plasmid screening

Plasmids were screening using the UltraCleanTM 6 Minute Mini Plasmid Prep Kit TM

(MoBio Laboratories, Inc., Solana Beach, CA). Briefly, 5mL of each culture was pelleted

in a 2mL microfuge tube and all remaining liquid was removed. The cell pellet was

resuspended in 50µL Solution 1 containing RNase A before 100µL of Solution 2 was

added to lyse the cells using alkaline lysis. This mixture was then neutralized by adding

325µL Solution 3 and mixed by gentle inversion. This mixture was then centrifuged at

10, 000 x g for 5 minutes. The supernatant was transferred into a collection tube

containing a spin filter unit and centrifuged at 10, 000 x g for 30 seconds. The liquid in

the collection tube was discarded and the spin filter replaced in the same tube. The spin

filter was washed by adding 300µL of Solution 4, containing 50% ethanol and

centrifuged for 30 seconds. The spin filter was placed in a new microfuge tube and the

plasmid eluted by adding 50µL Solution 5 into the membrane of the spin filter and

centrifuging at 10, 000 x g. The eluted plasmid was screened for desired insert by

performing a routine PCR with primers specific to gene insert. These plasmids were then

used for making DNA standards for qPCR studies.

Chapter 2

39

2.4. Terminal dUTP-nick End Labeling (TUNEL) Assay

In cells undergoing apoptosis, endogenous endonucleases cleave DNA and generate

DNA fragments of 180-200bp fragments. Apoptotic cells were identified using a

DeadEndTM Colorimentric TUNEL System (Promega, Madison, WI), which is a non-

radioactive system that detects apoptotic cells by labeling fragmented DNA in situ.

For tissue culture, cells were first fixed in 10% buffered formalin for 25 minutes at room

temperature before washing with PBS and permeabilized with 0.2% (v/v) Triton X-100 in

PBS for 5 minutes. The slides were then washed in PBS and equilibrated with 100µL

Equilibration Buffer for 10 minutes. The area surrounding the cells were blot dry and

rTdT reaction mixture (98µL Equilibration Buffer, 1µL Biotinylated Nucleotide Mix and

1µL rTdT Enzyme per 100 µL) was added to the cells and incubated for an hour at 37oC

in a humidified chamber to allow end-labeling to occur. To ensure the cells did not dry

out and allow even distribution of the reaction mixture, sections were covered with

plastic coverslips. The reaction was terminated by immersing the slides in 2X SSC buffer

for 15 minutes. The slides were washed with PBS to remove unincorporated biotinylated

nucleotides. Slides were blocked for endogenous peroxidases by immersing in 0.3% (v/v)

hydrogen peroxide for 5 minutes followed by washing in PBS. Streptavidin HRP solution

was diluted 1:500 in PBS and added to each slide and incubated for 30 minutes. TUNEL-

positive cells were visualized using liquid DAB.

Chapter 2

40

The same method was used to detect apoptotic cells in paraffin embedded tissue sections

with the following exceptions. Tissue sections were fixed by immersing slides in 10%

buffered formalin for 15 minutes after paraffin was removed. Tissue sections were

permeabilized using Proteinase K digestion as described in Section 2.1.2. Sections were

then washed in PBS and refixed in 10% buffered formalin for 5 minutes before

equilibration.

2.5. Animal Studies

The animals used in experiments were 4-week old male C57Bl/6 mice weighing between

12-16g. All animal experiments were performed in a pathogen-free animal holding

facility in accordance with guidelines of the National Health and Medical Research

Council of Australia and approved by the University of Western Australia Animal Ethics

Committee.

2.5.1. CDE Feeding

Mice were fed choline-deficient chow and 0.15% ethionine dissolved in drinking water

226. This diet was generally fed ad libitum for 14 days when maximal numbers of HPC

are present.

2.5.2. Euthanasia

Each mouse was anaesthetized with 10mg Ketamil (Troy Laboratories, New South

Wales, Australia) and 1mg Ilium Xylazil-20 (Troy Laboratories) in 0.43mL sterile saline.

Chapter 3

41

3. Interferon α Decreases Numbers of Hepatic

Progenitor Cells in Patients with Chronic Hepatitis C

3.1. Introduction The gold standard for treatment of chronic hepatitis C infection is IFNα in combination

with ribavirin. As mentioned in the general introduction (Section 1.9.1) IFNα treatment

decreases the risk of HCC development in chronic hepatitis C patients regardless of viral

clearance. An 8- to 11-year post-therapy study comprising 250 chronic hepatitis C

patients found that treatment with IFNα improved long-term outcome with respect to

progression to cirrhosis and development of HCC regardless of viral clearance 227. In a

randomised controlled trial of 90 patients with cirrhosis due to hepatitis C, Nishiguchi

and colleagues demonstrated that IFNα therapy significantly reduced progression to HCC

228. Similarly, Benvegnù and colleagues performed a retrospective analysis of the effects

of IFNα therapy on the clinical course and development of HCC on a cohort of 189

patients and found that treatment with IFNα was an independent variable associated with

reduced risk of disease progression and of HCC development 229. These studies of

chronically infected individuals have established that IFNα treatment can indeed reduce

the risk of HCC development regardless of viral clearance 79,80,82,83,228,230.

Response to antiviral therapy is defined by the normalization of serum alanine

transaminase (biochemical response) and by the disappearance of HCV RNA from

patient serum (virological response). A response at the completion of a course of

Chapter 3

42

treatment is defined as an end of treatment response (ETR), however the goal is to

achieve a sustained response (SR). A sustain virological response (SVR) is defined as

undetectable HCV RNA at six months following completion of treatment. Patients who

develop abnormal ALT values or detectable serum HCV RNA following an ETR are said

to have relapsed. Finally, non-response occurs when ALT normalises but later becomes

abnormal while still on treatment, or when viral RNA remains detectable throughout

treatment 231

Several studies have shown that the numbers of HPC vary with necroinflammatory

activity observed in chronic viral hepatitis 69,71,153. While the co-existence of HPC with

liver cancer has long been observed 70,94,155,232-234, it was not until recently that conclusive

data explaining the relationship between HPC and neoplasia was made available. As

discussed in Chapter 1, Shachaf and colleagues used conditional hepatic expression of the

c-myc proto-oncogene to clearly define the precursor-product relationship between HPC

and HCC 115. Transgenic mice which over-expressed c-myc developed tumors and these

tumors regressed en masse when c-myc was inactivated, differentiating into hepatocytes

and biliary cells in a manner similar to the previously observed differentiation of HPC

into these cell types. This differentiation was accompanied by a loss of AFP expression

and increase in expression of mature hepatocyte and biliary cell markers. HPC are

identified on the basis of typical histological appearance (small cells with oval nuclei)

combined with appropriate immunohistochemical makers summarized in Table 3.1. To

date, the mechanism of risk reduction for HCC afforded by IFNα has been unclear. It was

previously thought that IFNα caused a biochemical response in the non-virological

Chapter 3

43

responders, causing a reduction in ALT levels, and through this means, reduced liver

injury 235-237. Based on the precursor-product relationship between HPC and HCC, we

hypothesize that IFNα also exerts a direct effect on the HPC population – the population

of cells, which are the proposed targets for malignant transformation. Specifically,

IFNα may reduce the risk of HCC development by reducing the numbers of HPC either

through inhibition of proliferation, induction of apoptosis, and/or induction of

differentiation.

The aim of this study was to determine the effect of IFNα-based therapy on numbers of

HPC in subjects undergoing therapy for chronic hepatitis C.

Chapter 3

44

Table 3.1. Summary of immunohistochemical and cell surface markers expressed by HPC Marker Progenitor cells Hepatocytes Bile Ducts Haematopoietic cells References

Albumin + + - - 45,59,238

AFP + Fetal - - 45,59,238

π-GST + Fetal + - 239

M2-PK + Fetal + + 69

CK8 + + + - 45,238,240

CK14 +/- - - - 59,60,240

CK18 + + + - 45,238,240

CK19 +/- - + - 69,240

OV-6 + - + - 44

OC.2

OC.3 + +

241,242

A6 + - + - 243

Thy-1 + - - + 59,173,244

c-kit + - - + 48,245,246

Sca-1 + - - + 62,244

CD34 + - - + 48,238,244,247

AFP: Alphafetoprotein; CK: Cytokeratin; π-GST: pi-Glutathione-S-Transferase; M2-PK: M2-Pyruvate Kinase; OV: Oval Cell Marker

Chapter 3

45

3.2. Materials and Methods

3.2.1. Tissue Samples

Liver biopsies collected before and after interferon-based treatment were available from

16 randomly selected, non-responding individuals, with chronic hepatitis C

(predominantly genotype 1). The time interval between pre- and post-treatment biopsies

was 18 months; patients were treated for 48 weeks and the post-treatment biopsy was

performed at 6 months after end of treatment. Initial METAVIR scores ranged from 0 to

4. Informed consent was obtained for the use of these biopsies for research purposes, and

the study was approved by the Human Research Ethics Committee, Fremantle Hospital,

Fremantle, Western Australia. Standard histological examination was undertaken using

H&E and Masson’s trichrome staining for assessment of fibrosis. Of 16 patients, only 3

responded to IFN-α with virological clearance as detailed in Table 3.2.

Chapter 3

46

Table 3.2. Liver Biopsy Samples From Chronic Hepatitis C Patients

Patient # Age (Years) Gender

METAVIR Score

(Before)

METAVIR Score

(After) Virological response

1 54 Female 1 1 Non responder

2 50 Male 4 4 Non responder



5 45 Male 1 1 Responder

6 43 Female 0 1 Responder







13 38 Male 1 1 Responder




Chapter 3

47

3.2.2. Immunohistochemical Staining of Hepatic Progenitor Cells and

Inflammatory Infiltrate

All sections were antigen retrieved in citrate buffer as described in Section 2.1.2. Sections

were blocked for endogenous peroxidases and endogenous biotin using a biotin blocking

system (Dako, Carpinteria, CA) according to the manufacturer’s instructions. Sections

were then blocked for non-specific antigens using serum-free protein block (Dako).

Primary antibodies against c-kit (Dako, Golstrup, Denmark; 1:200), CK19 (Dako, 1:100),

Leukocyte Common Antigen (LCA, Dako, 1:200) and π-GST (Novocastra Laboratories,

Newcastle upon Tyne, UK, 1:400) were applied to tissue sections with dilutions as

described in Table 3.3 below, and incubated overnight at 4oC. Detection was performed

using the Universal LSAB®2 Kit (Dako) and liquid DAB (Dako). HPC were identified as

small cells with ovoid nuclei and scant cytoplasm that were immunoreactive for c-kit,

CK19 or π-GST. Inflammatory cells were identified as cells that were immunoreactive

for LCA.

Chapter 3

48

3.2.3. Statistical Analysis

Data were reported as the mean + SEM. Comparisons between groups were performed

using unpaired t-test or Fishers exact test (GraphPad Prism version 4.03 for Windows,

San Diego, CA). Means were deemed to be different for p< 0.05. Non-parametric data

are reported as median values and compared using the Mann-Whitney U test (GraphPad

Prism).

Chapter 3

49

3.3. Results

3.3.1. Interferon α Treatment Reduced Hepatic Progenitor Cell Numbers as

Demonstrated by Immunohistochemical Staining with c-kit, CK19 and π-

GST

HPC were identified as small cells with ovoid nuclei and scant cytoplasm that were

immunoreactive for CK19, π-GST or c-kit located in the periportal regions of the liver.

Treatment with IFNα significantly reduced the numbers of c-kit-positive HPC in chronic

hepatitis C patients (*p<0.05, A). However, IFNα treatment did not significantly affect

the CK19- or π-GST-positive HPC population in the liver.

Chapter 3

50

Figure 3.1. Change in HPC numbers determined by immunohistochemical staining. Following treatment, c-kit-positive cells decreased by 50% in 13 out of 16 patients (*p<0.05, A). However it did not cause any significant changes in the CK19 (B) or π−GST-(C) positive cells.

C Pre-treatment Post-treatment

0123456789

1011121314

% P

ositi

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ells

B Pre-treatment Post-treatment0123456789

% P

ositi

ve C

ells

A Pre-treatment Post-treatment0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

% P

ositi

ve C

ells

Chapter 3

51

3.3.2. Interferon-α Treatment Did Not Affect Inflammatory Infiltration

Inflammatory cells were identified as cells immunoreactive for LCA antibody. Liver

biopsy sections were examined for numbers of LCA positive cells to determine the effect

of IFNα on liver inflammation. No relationship was observed between treatment and

numbers of LCA positive cells (Figure 3.2).

Figure 3.2. Identification of inflammatory cells using LCA immunohistochemical staining. Treatment with IFNα did not appear to change inflammatory status of chronic hepatitis C patients.

Pre-treatment Post-treatment0

5

10

15

20

25

30

35

% P

ositi

ve C

ells

Chapter 3

52

3.4. Discussion

It has been suggested that IFNα therapy reduces the risk of HCC development in chronic

hepatitis C patients independent of its anti-viral effects. Additionally, there is mounting

evidence suggesting that at least a subset of liver tumors arise by malignant

transformation of HPC. As such, we investigated the effects of interferon treatment in

human subjects with chronic hepatitis C to determine if IFNα treatment modulates the

numbers of these cells.

IFNα therapy only reduced the c-kit-positive population of HPC. The marked change in

c-kit-positive cells after treatment demonstrates that this is a population of cells most

susceptible to the effects of IFNα, presumably because they represent the least

differentiated of the three populations studied. As a marker for early stages of hepatic

stem cell commitment, c-kit is expressed by the less differentiated counterparts of the

HPC compartment and is abruptly switched off as the cell differentiates 245,248,249. HPC

then begin to express more mature markers such as CK19 and π-GST 48,250. The

progenitor cell marker, π-GST, is found in fetal hepatocytes and duct-like progenitor cells

while CK19 is expressed by biliary epithelium and progenitor cells 131,239,250. Hence the

reduction in c-kit-positive population could mean one of two things: (i) a decrease in c-

kit-positive cells through inhibition of proliferation or apoptosis of the c-kit-positive

cells, or (ii) an induction of c-kit-positive cells to become more mature and hence stop

expressing c-kit to express more mature markers such as CK19 and π-GST. Additionally,

Chapter 3

53

c-kit has been claimed to stain more specifically for HPC compared to CK19 and π-GST

250, implying that IFNα effects are specific to the HPC population.

The presence of c-kit-positive cells has been reported in many cases of liver cancer

arising from a variety of underlying causes 97,251,252. Aberrant activation of c-kit and or its

ligand, stem cell factor (SCF) has been reported in several human malignant cells 253-257.

Bellone and colleagues demonstrated the multiple roles of c-kit activation in support of

malignant phenotype of colon carcinoma cells by enhancing survival, growth, migration

and invasive potential 254, suggesting that c-kit plays a crucial role in tumorigenesis, and

should not be merely compartmentalized as a marker for hepatic progenitor cells.

Treatment with pegylated interferon alpha 2B reduced the numbers of c-kit positive

intermediate hepatobiliary cells in 14 out of 16 non-responding subjects with chronic

hepatitis C. C-kit is a well-described marker for HPC 29,232,250, and has been previously

studied in human liver biopsies from patients with viral hepatitis, both with and without

HCC 250,252. Furthermore, we have recently shown that numbers of c-kit positive cells in

patients with chronic hepatitis B correlate with disease severity (Knight, B. et al,

submitted). Selective targeting of the c-kit-expressing cell population has also been

shown to offer potential benefits in the treatment of HCC 258. Our data suggest that the

anti-tumorigenic effects of interferon therapy in patients with hepatitis C may be

mediated by selective reduction of the c-kit expressing progenitor cell pool.

Chapter 3

54

The stem cell factor receptor, c-kit, is a proto-oncogene that encodes for a transmembrane

tyrosine kinase receptor structurally similar to PDGF and CSF-1 receptors 259,260. There is

an induction of SCF during early activation of oval cells by 2-AAF/PH 245. The receptor-

ligand complex (SCF/c-kit) play an important role in oval cell induction 248. Experiments

on c-kit tyrosine kinase (KIT) activity in Ws/Ws mice show that the SCF/c-kit system is

vital in the appearance of HPC either by regulating the numbers of HPC or by

committing HPC to differentiate 248.

Kinase inhibitor therapy has been in the limelight in recent years. Gleevec STI571, an

imatinib mesylate, is a tyrosine kinase inhibitor of c-kit first reported to cure HCC in

2004 258. Since then tyrosine kinases such as SCF/c-kit have been viewed as targets for

cancer therapy and this area of research extensively reviewed 261-264. More recently,

Gleevec has been used in a Phase II clinical trial of patients with advanced HCC 265. In

the small group of non-PDGR- and non-ckit-expressing HCC, the tyrosine kinase

inhibitor appeared to have no therapeutic effect – confirming that the application of

imantinib is limited to HCC expressing these tyrosine kinases. Other tyrosine kinase

inhibitors such as gefitinib (Iressa TM, ZD1839) targeting the tyrosine kinase activity of

the epidermal growth factor receptor (EGFR), have been used for the treatment of non-

small cell lung cancer, while trastuzumab (Herceptin®) is used for the treatment of

metastatic breast cancer due to its targeting of the Her2/neu receptor.

The concept of cancer stem cells has become widely accepted and hence differentiation

therapy has also become a subject of recent research focus. The general approach to

Chapter 3

55

differentiation therapy is based on the assumption that cancer stem cells can be

encouraged to undergo differentiation, leading to tumor reprogramming and resulting in

an induction of terminal differentiation and eventual loss of proliferative capacity 266-272.

A prominent figure in the field of HPC research has reviewed this subject extensively

273,274.

In conclusion, IFNα treatment of chronic hepatitis C may reduce the risk of liver cancer

development through control of the HPC population. These cells have been suggested to

be targets of malignant transformation. Of the three markers used in this study, c-kit

identifies the subpopulation of these cells most susceptible to the effect of IFNα possibly

because they are the less differentiated compared to CK19- or π-GST-positive cells and

have greater plasticity. However, the mechanisms by which IFNα treatment reduces HPC

numbers remain to be determined. The experiments described in Chapter 4 seek to

understand these mechanisms by studying the effects of IFNα on HPC in vitro.

Chapter 4

56

4. Effect of Interferon α on Proliferation,

Differentiation and Apoptosis of Hepatic Progenitor

Cells in vitro

4.1. Introduction

In the previous chapter, treatment of chronic hepatitis C infection with IFNα reduced the

numbers of hepatic progenitor cells which are the suggested targets of malignant

transformation leading to the development of HCC. However, the mechanism(s)

responsible for the reduction in the numbers of hepatic progenitor cells was not

elucidated. It is possible that IFNα produced these effects through modulation of

proliferation, apoptosis or differentiation. Previous studies of the effects of IFNα on these

cellular processes in HCC cell lines have demonstrated that IFNα signaling is mediated

by the Jak-STAT pathway. Radaeva and colleagues studied the molecular events

governing the effects of IFNα on hepatocytes and HCC cell lines of man and mouse 275.

They elucidated that IFNα activates multiple STATs including STAT-1, STAT-2, STAT-

3 and STAT-5. Using microarray analysis of 12, 000 genes in primary human hepatocytes

and HepG2 cells it has been determined that IFNα activates a variety of pro-apoptotic

and anti-tumor genes. This includes STAT-1, which regulates apoptosis through

maintenance of low constitutive levels of caspases 276, and inhibition of NK-κB activity

277 as well as IRF-1, a potent tumor suppressor 278,279. Given a plausible signalling basis

Chapter 4

57

for the effects of IFNα on cellular proliferation, differentiation and apoptosis, the aim of

this chapter was to determine if IFNα influences these processes in HPC in vitro.

Chapter 4

58

4.2. Materials and Methods

4.2.1. Stimulation of PIL-2 and PIL-4 with Interferon α

The HPC used in this study were PIL (p53-null immortalized liver) cells derived from

HPC in CDE-fed p53 -/- mice. Two distinct cell lines were used; PIL-2 and PIL-4. The

former exhibits tumorigenicity and is less heterogenous than the latter 93. Cells were

cultured and maintained as described in Section 2.1. The cells were washed with a mild

acid buffer and serum-starved as detailed in Section 2.2 to remove ligands before

overnight incubation with serum-free media for synchronization prior to treatment with

IFNα.

Chapter 4

59

4.2.2. Determining the Effect of Interferon α on Mitochondrial Activity Using

MTT Assay

The effect of IFNα on mitochondrial activity of PIL-2 and PIL-4 cells was determined by

means of an MTT (3-[4, 5- dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide)

assay. This is a colorimetric method for measuring the number of viable cells in a

proliferation or cytotoxicity assay. The yellow tetrazolium MTT salt is reduced by

metabolically active cells, by the action of dehydrogenase enzymes, to generate reducing

equivalents such as NADH and NADPH, which produces a purple formazan that can be

quantified via spectrophotometry. The assay indirectly measures cell proliferation rate

and reduction in cell viability.

Approximately 2, 000 cells were seeded in each well of a 96-well, flat-bottomed plate.

These cells were stimulated with IFNα after washing with an acid buffer and serum

starved as described in Section 4.2.1. After 24 hours of IFNα treatment, the MTT

proliferation assay was performed. Briefly, 20 µL of MTT (5mg/mL, Sigma) was added

to each well followed by incubation at 37oC for 4 hrs. After incubation, 100 µL of

solubilization buffer (0.5M SDS, 50% v/v DMF, pH 4.7 with glacial acetic acid) was

added to each well and absorbance readings were taken at 550 nm. Standard curves were

constructed with known numbers of cells in each well for verification of optimum

absorbance range and readings were normalized against untreated cells of the same

seeding density.

Chapter 4

60

4.2.3. Determining the Effect of Interferon α on Proliferation using

Proliferating Cell Nuclear Antigen Staining

PIL-2 and PIL-4 cells were stained for proliferating nuclear antigen (PCNA) to assess

proliferative status of the cells using a previously described method 232. In brief, cells

were stimulated with 250IU/mL IFNα in Williams E media for 24 hours and fixed using

4% (v/v) buffered formalin for 8 minutes and 99% ethanol for 2 minutes. Cells were then

washed in phosphate buffered saline (PBS) and blocked for endogenous peroxidases with

3% (v/v) hydrogen peroxide for 1 minute, followed by a biotin blocking step using a

biotin blocking kit (Dako, Carpinteria, CA, USA) where cells were incubated with 0.1%

avidin and 0.01% biotin for 10 minutes each. Cells were also blocked with a serum-free

protein block (Dako) for 10 minutes before incubating with the mouse monoclonal anti-

PCNA primary antibody diluted in PBS at 1:200, for 1 hour at 37oC. Following this, cells

were washed in PBS and incubated with biotinylated anti-mouse secondary antibody and

streptavidin-labeled alkaline phosphatase in Universal LSAB®2 Kit (Dako) for 30

minutes each. Cells positive for PCNA-staining were visualized using the DAB

chromagen kit (Dako).

Chapter 4

61

4.2.4. Determining the Effect of Interferon α on Cyclin D1 Expression

PIL-2 and PIL-4 cells were seeded in 6-well plates at a density of 2, 000 cells per well

and grown to 70% confluence before treating with 250 IU/mL IFNα for 24 hours. The

changes in mRNA levels of cyclin D1 were measured using qPCR. Briefly, RNA was

isolated from stimulated and unstimulated PIL cells as described in Section 2.3, and

DNAse treated as described in Section 2.5, while cDNA synthesis was performed as

described in Section 2.6. Each qPCR reaction contained 1X reaction buffer (Fisher

Biotech, Perth, WA, Australia), 1.25mM MgCL2 (Fisher Biotech), 0.25mM dNTPs

(Fisher Biotech), 2 units of Platinum Taq (Invitrogen, Calsbad, CA, USA) and 2µL of

cDNA. The primers used for this were: (forward) 5’-CTG ACA CCA ATC TCC TCA

ACG-3’ and (reverse) 5’-GTC GGC CAG GTT CCA CTT GAG-3’. The mRNA were

amplified and measured against serially diluted known concentrations of DNA standards

bearing the same sequence, using the RotorgeneTM 3000 (Corbett Research, Sydney,

NSW, Australia), where fluorescence threshold was set to 10% and gain was set between

7 and 10 for each run. Amplicons were compared against known standards and expressed

as number of copies per µL and then normalized against β-actin expression. DNA

standards were made by cloning PCR products using the pGEM®T Easy Vector System

(Promega, Madison, WI, USA) and screened using UltraCleanTM 6 Minute Plasmid Prep

Kit (MoBio Laboratories Inc., Solana Beach, CA, USA) as detailed in Sections 2.7 and

2.8 respectively.

Chapter 4

62

4.2.5. Detecting Apoptotic Cells Using Terminal dUTP Nick End Labeling

(TUNEL) Assay

PIL-2 and PIL-4 cells were seeded in 4-chambered slides (BD Falcon) at a density of 200

cells per chamber. After allowing for adherence, cells were serum-starved overnight prior

to 3 days stimulation with 250 IU/mL IFNα. Apoptotic cells were detected using TUNEL

assay (Promega, Madison, WI, USA) according to manufacturer’s instructions. Detailed

methodology was described in Section 2.10.

4.2.6. Determining Changes in Differentiation Status Using Quantitative PCR The changes in expression of various markers correlating to specific stages of maturation

and lineages was measured using qPCR. PIL-2 and PIL-4 were grown in Williams E

media containing 250IU/mL IFNα. Isolation of RNA, synthesis of cDNA and reaction

mixtures were described in Section 4.2.4. Primers for genes analysed are detailed in

Table 4.1. The annealing temperature for all the primers used in this assay was 60oC.

Quantitation of gene expression was performed as described in Section 4.2.4.

Chapter 4

63

Table 4.1. Primers for target gene sequences analyzed by quantitative PCR TARGET GENE PRIMER SEQUENCE PRODUCT

SIZE

SOURCE

Albumin 5’ CTT AAA CCG ATG GGC GAT CTC ACT

3’ CCC CAC TAG CCT CTG GCA AAA T

130 bp 148

AFP 5’ TCG TAT TCC AAC AGG AGG

3’ AGG CTT TTG CTT CAC CAG

176 bp 148

CK19 5’ CAT GGT TCT TCT TCA GGT AGG C

3’ GCT GCA GAT GCA TTC AGA ACC

177 bp Strick-Marchand, H. Pasteur Institute, Paris

Thy-1 5’ AAC CAA AAC CTT CGC CTG GAC

3’ AAG CTC ACA AAA GTA GTC GCC C

231 bp 280

G6Pse 5’ CGC CTT CTA TGT CCT CTT TCC C

3’ GTT TCA GCC ACA GCA ATG CC

459 bp 148

Chapter 4

64

4.3. Results

4.3.1. Interferon α Inhibits Proliferation in Both PIL-2 and PIL-4

IFNα exerted an inhibitory effect on mitochondrial activity of both PIL-2 and PIL-4.

Increasing concentrations of IFNα resulted in a dose-dependent reduction in cell viability

as shown by MTT assay (Figure 4.1) where LD50 was determined to be approximately

250 IU/mL.

Figure 4.1. MTT-based proliferation assay. Effect of IFNα (250 IU/mL) on proliferation of PIL-2 cells (A) and PIL-4 cells (B) after 24 hours in culture.

1 2 4 8 16 32 64 128 256 512 10240.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.9

IFNα (U/mL)

Cel

l via

bilit

y no

rmal

ised

aga

inst

neg

ativ

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ntro

l

2 4 8 16 32 64 128 256 512 1024

-0.2-0.10.00.10.20.30.40.50.60.70.80.91.01.11.21.3

IFNα (U/mL)

Cel

l via

bilit

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rmal

ised

aga

inst

neg

ativ

e co

ntro

l

A

B

Chapter 4

65

This reduction in mitochondrial activity was confirmed to be a result of decrease in

proliferation as seen in reduced numbers of PCNA-positive cells (Figure 4.2).

IFNα Untreated0

10

20

30

40

50

60

70

*

p<0.001

**

% P

CN

A po

sitiv

e ce

lls

IFNα Untreated0

25

50

75

100

*

p < 0.001

**

% P

CN

A p

ositi

ve c

ells

Figure 4.2. Treatment with IFNα changes proliferation status of PIL-2 and PIL-4 cells. Treatment with IFNα caused a significant decrease in PCNA-positive PIL-2 cells (A**p<0.001). Representative photographs of PCNA staining of PIL-2 cells grown in control media and media supplemented with 250 IU/mL IFNα are shown in B and C respectively. Similarly, IFNα treatment resulted in a decrease in PCNA-positive PIL-4 cells (D. **p<0.001). Representative photographs of PCNA staining of PIL-4 cells grown in control media and media supplemented with 250 IU/mL IFNα are shown in E and F respectively.

A

C

D

F

E

Chapter 4

66

4.3.2. Interferon α Reduces Cyclin D1 Expression in PIL-2 But Not PIL-4

Cyclin D1 expression changed following 24 hours treatment of PIL-2 and PIL-4 cells

with IFNα. Treatment of PIL-2 cells with IFNα resulted in reduced expression of cyclin

D1. However, cyclin D1 expression was not affected by IFNα in the PIL-4 cells (Figure

4.3).

Figure 4.3. Changes in cyclin D1 expression in PIL-2 and PIL-4 cells following 24 hour treatment with IFNα. IFNα resulted in a greater than 50% reduction in Cyclin D1 expression in PIL-2 cells (A, **p<0.001). No significant effect of IFNα on the expression of Cyclin D1 by PIL-4 cells was observed (B). Note: mRNA levels of cyclin D1 were normalized against mRNA levels of β-actin.

A B

untreated IFNα0

2

4

6

** **

Cyc

lin D

1 m

RN

A le

vels

untreated IFNα0.0

1.5

3.0

4.5C

yclin

D1

mR

NA

leve

ls

Chapter 4

67

4.3.3. Interferon α Causes Apoptosis in Both PIL-2 and PIL-4

IFNα caused apoptosis in both PIL-2 and PIL-4 cells as demonstrated by TUNEL

staining; there was a 7-fold increase in the percentage of apoptotic PIL-2 cells (Figure

4.4.A, ***p<0.0001) and a 14-fold increase in the percentage of apoptotic PIL-4 cells

(Figure 4.4.B, ***p<0.0001) following treatment with IFNα.

Figure 4.4. Effect of IFNα on apoptosis of PIL cells. The percentage of apoptotic cells significantly increased in both PIL cell lines following administration of IFNα. A 7-fold increase was seen in PIL-2 cells (A, ***p<0.0001) and 14-fold increase was seen in PIL-4 cells (B, ***p<0.0001)

untreated IFNα0

10

20

30

40

50

% T

UN

EL-p

ositi

ve c

ells

***

untreated IFNα0

10

20

30

40

50

% T

UN

EL-p

ositi

ve c

ells ***

A B

Chapter 4

68

4.3.4. Interferon α Causes PIL-2 and PIL-4 Cells to Differentiate

To determine if IFNα treatment of oval cell lines was associated with an alteration in

maturation state, the change in relative expression of key hepatic developmental markers

was measured in vitro following treatment with IFNα. Following 8 days exposure to

pegylated IFNα, albumin mRNA levels increased by 19-fold in PIL-2 cells (Figure 4.5A,

***p<0.001) while in PIL-4 cells the levels remained unchanged (Figure 4.5B). CK19

mRNA levels increased 3-fold following treatment in both cell lines (Figure 4.5C & D,

*p<0.05). PIL-2 and PIL-4 cells exhibited a decrease in AFP gene expression of 1- and

323-fold, respectively (Figure 4.5E & F, *p<0.05). G6Pase mRNA levels increased by

414-fold in PIL-4 cells after administration of IFNα (Figure 4.5H, *p<0.05) but remained

unaffected in PIL-2 cells. Thy-1 was only expressed in PIL-2 cells and its expression was

significantly reduced following administration of IFNα (Figure 4.5I, *p<0.05).

Chapter 4

69

Figure 4.5. Effects of IFNα on differentiation of PIL cells. Following 8 days treatment with 250U/mL IFNα, significant changes were detected in the expression of markers specific to various stages of maturation. There was an increase in the expression of the mature markers albumin in PIL-2 cells (A, ***p<0.001) but no significant effect on PIL-4 cells (B). Expression of CK19 increased in both cell lines following IFNα treatment (PIL-2 C & PIL-4 D, *p<0.05). Note: mRNA levels of hepatic maturation markers were normalized against mRNA levels of β-actin.

D

untreated IFNα 0

5.0×10-8

1.0×10-7

1.5×10-7

2.0×10-7

2.5×10-7

3.0×10-7 ***

Alb

umin

mR

NA

leve

ls

A

untreated IFNα0

7.0×10-6

1.4×10-5 *

CK

19m

RN

A le

vels

B

C

untreated IFNα0

3.0×10-5

6.0×10-5

9.0×10-5

Alb

umin

mR

NA

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ls

untreated IFNα0

2.0×10-4

4.0×10-4

6.0×10-4

8.0×10-4

1.0×10-3

1.2×10-3

*

CK

19m

RN

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Chapter 4

70

Figure 4.5. Effects of IFNα on differentiation of PIL cells (continued). Treatment with IFNα caused a decrease in the expression of the less matured marker AFP in both cell lines (PIL-2 E & PIL-4 F, *p<0.05). While no significant change was detected in the expression of G6Pase by PIL-2 cells (G), IFNα significantly increased its expression by PIL-4 cells (H, *p<0.05). Finally, IFNα reduced expression of Thy-1 in PIL-2 cells (I, *p<0.05), while PIL-4 cells did not express this marker. Note: mRNA levels of hepatic maturation markers were normalized against mRNA levels of β-actin.

untreated IFNα0

3.0×10-4

6.0×10-4

9.0×10-4

*

Thy-

1m

RN

A le

vels

untreated IFNα0

2.5×10-6

5.0×10-6

7.5×10-6

1.0×10-5

1.2×10-5

1.5×10-5

1.7×10-5

*

AFP

mR

NA

leve

ls

untreated IFNα0

1.0×10-6

2.0×10-6

3.0×10-6

4.0×10-6

5.0×10-6

*A

FP m

RN

A le

vels

E F

untreated IFN α0

1.0×10-5

2.0×10-5

3.0×10-5

4.0×10-5

5.0×10-5

G6P

ase

mR

NA

leve

ls

G

untreated IFNα0

2.0×10-3

4.0×10-3

6.0×10-3

8.0×10-3

1.0×10-2

1.2×10-2 *

G6P

ase

mR

NA

leve

ls

H

I

Chapter 4

71

4.3.5. Interferon α Did Not Affect the Morphology of PIL-2 and PIL-4

Treatment of PIL-2 with IFNα did not result in any identifiable changes in the

morphology of the cells after 8 days culture in Williams E medium containing 250 IU/mL

IFNα as illustrated in Figure 4.6.

Figure 4.6. Treatment of PIL-2 cells with IFNα does not result in morphological changes after 8 days of treatment. Figure 4.6A is a representative photograph of PIL-2 cells after being cultured in medium containing 250 IU/mL IFNα for 6 days at 40X magnification. Figure 4.7B shows PIL-2 cells after being cultured in control Williams E medium for 6 days. Figures 4.6C and D are photographs of PIL-2 cells after 8 days exposure to either IFNα or control medium.

B

D

A IFNα 6 days

C IFNα 8 days

Untreated 6 days

Untreated 8 days

Chapter 4

72

Similarly, IFNα did not induce obvious morphological changes in the PIL-4 cells

following 6 or 8 days of treatment (Figure 4.7).

Figure 4.7. Treatment of PIL-4 cells with IFNα does not result in morphological changes after 8 days of treatment. Figure 4.7A is a representative photograph of PIL-4 cells after being cultured in media containing 250 IU/mL IFNα for 6 days at 40X magnification. Figure 4.7B shows PIL-4 cells after being cultured in control Williams E media for 6 days. Figures 4.7C and D show PIL-4 cells after 8 days in culture either in the presence or absence of IFNα.

A IFNα 6 days B Untreated 6 days

C IFNα 8 days D Untreated 8 days

Chapter 4

73

4.3. Discussion To elucidate the mechanism by which IFNα treatment reduced the numbers of HPC

present in the chronic hepatitis C patients, we examined the effects of IFNα on the

proliferation, apoptosis and differentiation of two well characterized murine HPC cell

lines. The results suggest that IFNα may exert direct effects on HPC; reducing their rate

of cell growth, as well as stimulating them to undergo apoptosis.

In the current study, a dose-dependent inhibitory effect on proliferation was observed in

both cell lines accompanied by a significant reduction of cyclin D1 mRNA levels in one

cell line. This is in accordance with the observed effects of IFNα on human HCC-derived

cell lines, in which IFNα interfered with cell cycling causing blockage at G2/M phase as

well as at the G1 phase 183 and delayed S-phase progression through inhibition of cyclin-

dependent kinases 181.

The current study shows that IFNα causes HPC to undergo apoptosis in vitro as

demonstrated by TUNEL-staining. Other studies of the effects of IFNα on malignant cell

lines have suggested that this apoptosis is caspase-dependent, implicating the role of the

Fas/Fas ligand, as well the significant loss of membrane potential in the IFNα affected

cell 197. IFNα has been shown to sensitize hepatoma cells to TRAIL-induced apoptosis

via the upregulation of DR5, a death receptor of TRAIL; accompanied by the

downregulation of NF-κB, a transcription factor which plays a key role in cell survival

184.

Chapter 4

74

Interestingly, after treating PIL-2 and PIL-4 cells with IFNα for 8 days, significant

changes were observed in the expression of various marker genes corresponding to

different stages of HPC maturity and commitment to lineage. The expression of the

immature cell markers AFP and Thy-1 was dramatically reduced following culture in the

presence of IFNα, suggesting that their maturation was cytokine-stimulated. These

findings were corroborated by the increased expression of the mature biliary and

hepatocytic markers: CK19, albumin and G6Pase, in one or both of the cell lines. This

suggests that in addition to its growth-modulatory effects, IFNα may promote the

maturation of HPC.

Activation of the HPC compartments has been demonstrated in hepatocarcinogenic

conditions such as chronic viral and alcoholic hepatitis. Additionally, HCC expresses

HPC markers including AFP, CK7, CK19, CK4 and hoxa-13 mRNA. Thus the precursor-

product relationship between HPC and HCC cannot be ignored. Findings from this study

demonstrate that IFNα may be an effective agent for control of the HPC population.

When combined with the findings reported in Chapter 3, it is proposed that IFNα may

reduce the risk of HCC development in conditions such as chronic hepatitis C by

regulating numbers of target cells via modulation of apoptosis, proliferation and

differentiation.

Chapter 5

75

5. Effects of Interferon α on Hepatic Progenitor

Cells in vivo

5.1. Introduction

In Chapters 3 and 4, we reported that IFNα caused a reduction in HPC numbers in both

in vitro experiments as well as in the chronic hepatitis C patients. It is hence important to

validate these results with an animal model of progenitor cell activation. As previously

mentioned in Chapter 1, these cells are not usually seen in healthy livers, but are detected

only during instances of chronic liver injury and their numbers vary according to disease

severity.

Previous studies by our group and others have suggested that there is an orchestra of

cytokines and growth factors that play vital roles in activation and expansion of these

progenitor cells including, but not limited to, lymphotoxin-β (LTβ), tumor necrosis

factor-α (TNFα), transforming growth factor-β (TGFβ), interferon-γ (IFNγ) and

interleukin-6 (IL-6) 154,167,168,224,281,282. Therefore, we hypothesized that IFNα reduces

HPC numbers through alteration of these cytokines and growth factors.

In this study, C57Bl/6 mice were placed on the CDE diet to induce liver injury. The CDE

diet is a carcinogenic diet that causes chronic liver injury and is optimal for the

stimulation of HPC response. It was first developed by Shinozuka and colleagues in 1978

Chapter 5

76

for the rat model of hepatocarcinogenesis 283. The diet has since undergone numerous

modifications for various hepatocarcinogenic regimes. The diet used in this study consists

of choline deficient chow with ethionine supplemented in drinking water fed ad libitum.

The CDE diet is comprised of 0.165% ethionine dissolved in water and choline-deficient

chow which limits the induction of liver drug-metabolizing enzymes and hence leads to

alterations in metabolism of ethionine 284,285. DL-Ethionine is a well-known

hepatocarcinogen, and an antagonist of methionine; hence inhibitor of choline

biosynthesis 286. HPC numbers begin to increase rapidly from day 3 to day 14 of the

regime, after which their numbers plateau 154.

The aims of this study were to:

(i) confirm that IFNα reduced the numbers of HPC in vivo using a CDE murine

model of carcinogenesis; and

(ii) elucidate the effects of IFNα on cytokines and growth factors that have been

linked to activation and expansion of the HPC compartment.

Chapter 5

77

5.2. Methods

5.2.1. Animal Studies

To determine the in vivo effects of IFNα on the HPC population, four-week old C57Bl/6

male mice weighing between 14 to 16g were placed on the choline-deficient ethionine-

supplemented (CDE) diet to induce proliferation of HPC. Animals were administered

either 105 units of pegylated IFNα-2B (PEGATRON® Shering-Plough) or saline

(placebo) intraperitoneally once every 3 days. Mice were sacrificed two weeks later.

Livers were perfused with saline, and portions of liver were sampled for histological

studies.

5.2.2. Immunohistochemical Staining for A6, M2-Pyruvate Kinase (MPK)

and c-kit

Formalin fixed tissues were used for all immunohistochemical studies except for A6-

staining, which required frozen sections. Sections for MPK and c-kit staining were

antigen retrieved in EDTA buffer as described in Section 2.1.2. All sections were blocked

for endogenous peroxidases followed by blocking for non-specific antigens using serum-

free protein block (Dako). Primary antibodies against A6 (1:30; A kind gift from Dr

Valentina Factor, National Cancer Institute, National Institute of Health, Bethesda), MPK

(1:500; Rockland, Gilbertsville, PA) and c-kit (1:200, Dako) were applied to tissue

sections and incubated overnight at 4oC and detected using biotinylated secondary

antibodies and liquid DAB (Dako). HPC were identified as small cells with ovoid nuclei

and scant cytoplasm that were immunoreactive against A6, MPK or c-kit. The number of

Chapter 5

78

cells was determined by counting 10 periportal non-overlapping fields at 400X

magnification. HPC were expressed as the average percentage of total hepatocytes per

field of view.

5.2.3. Determining Changes in Proliferative Status Using PCNA Staining

The proliferative status of the liver was determined using PCNA staining as described in

Section 4.2.3, with the exception of using deparaffinised tissue sections. PCNA-positive

cells in non-overlapping periportal regions were counted at 40X magnification and

expressed a percentage of hepatocytes in the field.

5.2.4. Determining Changes in Apoptotic Status Using TUNEL Staining

Cells at the end stage of apoptosis were identified using TUNEL staining as described in

Section 2.4. These cells in non-overlapping periportal regions were counted at 40X

magnification and expressed as a percentage of total number of hepatocytes in the field.

5.2.5. RNA Isolation from Mouse Liver Tissues

RNA was isolated from fresh mouse liver tissue as described in Section 2.3.2.

Chapter 5

79

5.2.6. Determing Effect of Interferon α on Cytokine Profile

Changes in cytokine profile brought about by IFNα treatment of the CDE mice was

analyzed using qPCR. As previously described in Section 2.3.1., isolated RNA was

DNAse treated and cDNA was synthesized as described in Section 2.3.3. and 2.3.4.

cDNA was then subjected to qPCR analysis as described in Section 4.2.4. Annealing

temperatures (Tm) and extension times optimized for qPCR are detailed in Table 5.1.

Table 5.1. Primers for cytokine profile analyzed by quantitative PCR NAME PRIMER SEQUENCE 5’ 3’ SIZE Tm/EXTENSION TIME

IFNγ F CTT CTT GGA TAT CTG GAG GAA CT

IFNγ R CTC AAA CTT GGA AAT ACT CAT GAA 213bp 60oC / 20 seconds

TGFβ F CAC TGA TAC GCC TGA GTG

TGFβ F GTG AGC GCT GAA TCG AAA 100bp 55oC / 20 seconds

LTβ F TCG GGT TGA GAA GAT CAT TGG

LTβ R GCT CGT GTA CCA TAA CGA CC 640bp 52oC / 1 minute

TNFα F CGG AGT CCG GGC AGG T

TNFα R GCT GGG TAG AGA ATG GAT GAA CA 74bp 55oC / 20 seconds

IL-6 F GAG GAT ACC ACT CCC AAC AGA CC

IL-6 R AAG TGC ATC ATC GTT GTT CAT ACA 141bp 55oC / 20 seconds

Chapter 5

80

5.2.7. Histological Grading of Fibrosis

Histological grading of fibrosis was performed using sirus red staining. Briefly, sections

were deparaffinized and stained by placing drops of sirius red stain on tissue sections for

5 minutes, blotting dry and immersing directly into 70% ethanol. Fibrosis scoring was

performed using the following criteria: 0 for normal liver histology, 1 for mild fibrosis in

the periportal regions, 2 for moderate fibrosis in the periportal regions, 3 for septal and

bridging fibrosis between portal tracts and 4 for cirrhotic bands linking portal tracts.

Difference in fibrotic score between the two groups was determined using chi-square

analysis with a confidence interval of 95%.

5.2.8. Immunohistochemical Staining for Activated Hepatic Stellate Cells

Immunohistochemical staining for α−smooth muscle actin (αSMA) was performed to

identify activated HSC using a mouse monoclonal raised against mouse αSMA (Sigma-

Aldrich) and diluted to a concentration of 1:1000, applied to tissue sections and incubated

for an hour at room temperature before exposing to biotinylated secondary antibodies and

liquid DAB (Dako). Numbers of αSMA-positive cells in non-overlapping periportal

regions were counted at 400X magnification and expressed as a percentage of

hepatocytes in the field.

Chapter 5

81

5.2.9. Immunohistochemical Staining for Phosphorylated STAT-3

Antibodies specific to STAT-3 phosphorylated at the Ser705 residue was used to detect

cells containing phospho-STAT-3. Phosphorylation of STAT-3 at the Ser705 residue

induces dimerization, nuclear translocation and DNA binding. The antibody raised

against mouse phospho-STAT-3 (Cell Signaling Technology, Danvers, MA) was diluted

1:200, and applied to tissue sections, incubated overnight at 4oC before exposing to

biotinylated secondary antibodies and detecting using liquid DAB (Dako). Numbers of

phospho-STAT-3-positive cells in non-overlapping periportal regions were counted at

40X magnification and expressed as a percentage of hepatocytes in the field.

Chapter 5

82

5.3. Results

5.3.1. Interferon α Reduces Numbers of Hepatic Progenitor Cells in the CDE

Mouse Model

Treatment with IFNα significantly reduced the numbers of HPC. Following two weeks of

CDE diet feeding when progenitor cells were at their highest, treatment with pegylated

IFNα-2B resulted in a 4-fold reduction in the number of A6-positive HPC cells present in

the liver, in comparison to animals which received placebo (*p<0.05, Figure 5.1A).

Placebo IFNα0.0

2.5

5.0

7.5

10.0

12.5

*

% A

6 po

sitiv

e ce

lls

Figure 5.1. Treatment with IFNα reduced numbers of A6-positive cells in mice placed on CDE diet. A6-positive cells decreased by approximately 4-fold in IFNα treated group compared to placebo (*p<0.05, A). Representative photographs taken of A6-positive progenitor cells in placebo and IFNα−treated animals (B & C).

C

B

A

Chapter 5

83

This change in numbers of A6-positive cells was accompanied by a one-third decrease of

MPK-positive HPC (*p<0.05, Figure 5.2A-C).

Placebo IFNα

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

*

% M

PK-p

ositi

ve o

val c

ells

Figure 5.2. Treatment with IFNα reduced numbers of MPK-positive cells in mice placed on CDE diet. MPK-positive cells decreased by approximately 30% in IFNα treated group compared to placebo (*p<0.05, A). Representative photographs taken of MPK-positive HPC cells in placebo and IFNα−treated animals (B & C).

C

B

A

Chapter 5

84

In order to draw parallel comparisons between the reduction in HPC numbers seen in the

IFNα-treated CDE model and IFNα−treated chronic hepatitis C patients, it was necessary

to include at least one marker that was common to both studies. For this reason, an

immunohistochemical study was also performed to determine the effect that

IFNα treatment had on the numbers of c-kit positive cells in the CDE model. We

observed that there was an approximate 80% reduction in c-kit positive cells in the IFNα-

treated group compared to placebo (**p<0.005, Figure 5.3A).

Placebo IFNα

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

**

% c

-kit-

posi

tive

cells

Figure 5.3. Treatment IFNα reduced numbers of c-kit-positive cells in mice placed on CDE diet. The proportion of c-kit-positive cells was reduced by almost 5-fold in the IFNα treated group compared to placebo (**p<0.005, A). Representative photographs taken of c-kit-positive HPC in placebo and IFNα−treated animals (B & C).

C

B

A

Chapter 5

85

5.3.2. Interferon α Exerts a Differential Effect on Proliferation of Hepatic

Progenitor Cells and Hepatocytes

PCNA staining was performed on tissue sections to confirm the anti-proliferative effect

of IFNα on HPC as seen with the in vitro data presented in Chapter 3. Treatment of mice

with IFNα significantly reduced the proportion of PCNA-positive progenitor cells

(*p<0.05, Figure 5.4A), while increasing the proportion of PCNA-positive hepatocytes

(***p<0.001, Figure 5.4B). Demonstrating that IFNα had a differential effect on the

proliferative status of the HPC and the hepatocytes.

Figure 5.4. Treatment of mice on CDE diet with IFNα reduces proliferative status of progenitor cells while it increases proliferative status of hepatocytes. IFNα treatment reduced the mean percentage of PCNA-positive HPC by 20% (*p<0.05, A), while almost doubling the percentage of PCNA-positive hepatocytes (***p<0.001, B). Representative photographs of PCNA-staining in IFNα (C) and placebo (D) samples. Hepatocytes indicated by red arrows and progenitor cells indicated by black arrows.

A

B D

CPlacebo IFNα

0.0

0.5

1.0

1.5

2.0

2.5

*

% P

CN

A p

ositi

ve p

roge

nito

r ce

lls

*

Placebo IFNα0

1

2

3***

% P

CN

A p

ositi

ve h

epat

ocyt

es

Chapter 5

86

5.3.3. Interferon α Treatment Does Not Induce Apoptosis

A TUNEL assay was performed in order to determine if the reduction in A6-, MPK- and

c-kit-positive cells in the IFNα treated mice could be attributed to apoptosis of HPC, and

also to validate the in vitro TUNEL results presented in Chapter 4. This assay stains cells

at the end stages of apoptosis. Apoptotic hepatocytes can be differentiated from apoptotic

progenitor cells based on morphological differences, hence this experiment served two

purposes:

(i) to determine if IFNα exerted an effect specific to the progenitor cell

population, and

(ii) to confirm the apoptotic effect seen in the in vitro studies.

A slight increase in numbers of TUNEL-positive progenitor cells was observed in the

IFNα treated group as compared to placebo (Figure 5.5A). Likewise, there was no

obvious difference in the numbers of TUNEL-positive hepatocytes between the IFNα

treated group and placebo (Figure 5.5B).

Chapter 5

87

Figure 5.5. Treatment with IFNα increases proportion of apoptotic progenitor cells but not hepatocytes. The mean percentage of TUNEL-positive hepatic progenitor cells increased slightly, from 1.1% to 1.6%, however this difference was not significant (A). There was no change in proportion of TUNEL-positive hepatocytes (B). Representative photographs of TUNEL-staining in IFNα (C) and placebo (D) samples. Hepatocytes indicated by red arrows and progenitor cells indicated by black arrows.

A

B D

C

Placebo IFNα0.00

0.25

0.50

0.75

1.00

1.25

% T

UN

EL-p

ositi

ve h

epat

ocyt

es

Placebo IFNα0.000.250.500.751.001.251.501.752.002.25

% T

UN

EL-p

ositi

ve p

roge

nito

r ce

lls

Chapter 5

88

5.3.4. Interferon α Treatment Changes Cytokine Profile of CDE-fed Mice

Treatment with IFNα changed the cytokine profiles of mice placed on CDE diet. Hepatic

levels of IFNγ and LTβ decreased dramatically in the IFNα treated mice. Levels of

IFNγ were virtually undetectable at the end of the experiment (*p<0.05, Figure 5.6 A),

while LTβ levels were more than 2-fold higher in the placebo group compared to the

IFNα treated group (*p<0.05, Figure 5.6 B). This was accompanied by a significant 6-

fold reduction in TGFβ levels in the treated group compared to placebo (*p<0.05, Figure

5.6 C). However, no significant changes were observed on hepatic levels of TNFα and

IL-6 (Figure5.6 D&E).

Chapter 5

89

A Placebo IFNα0.0

5.0×1011

1.0×1012

1.5×1012

2.0×1012

2.5×1012

*IFN

γm

RN

A le

vels

D Placebo IFNα0

10

20

30

40

TNFα

mR

NA

leve

ls

B Placebo IFNα0.00

0.05

0.10

0.15

0.20

*

LTβ

mR

NA

leve

ls

E Placebo IFNα0.0

1.0×1012

2.0×1012

3.0×1012

4.0×1012

IL-6

mR

NA

leve

ls

Placebo IFNα0

1e-005

2e-005

3e-005

*

TGF β

mR

NA

leve

ls

Figure 5.6. Treatment with IFNα cause changes in cytokine profile of mice place on CDE diet. Minimal amount of IFNγ is detected following treatment (*p<0.05, A). Levels of LTβ in the IFNα group were less than half of placebo (*p<0.05, B). IFNγ levels were 6 times lower in treated animals compared to placebo (*p<0.05, C). No significant changes in TNFα and IL-6 were observed (D&E). Note: mRNA levels of cytokines were normalized against mRNA levels of β-actin.

C

Chapter 5

90

5.3.5. Interferon α Does Not Significantly Affect Fibrotic Status of CDE-fed

Mice

In the IFNα treated group, there were 4 mice which had healthy livers, while 2 were

graded at stage 1 of fibrosis where mild periportal fibrosis was observed. In the placebo

group, there were 2 mice in each group – healthy liver, mild periportal fibrosis and

moderate periportal fibrosis (Table 5.2). There was no significant difference in the

fibrotic status of the two groups of animals as determined by chi-square analysis.

Table 5.2. Interferon α treatment does not significantly affect the stage of fibrosis in CDE-fed mice

IFNα Placebo

Stage 0 4 2

Stage 1 2 2

Stage 2 0 2 *Stage 0 – Healthy liver *Stage 1 – Mild fibrosis isolated to periportal regions *Stage 2 – Moderate fibrosis isolated to periportal regions

Figure 5.7. Representative photographs of sirius red staining for fibrosis staging. There was no discernible difference between fibrotic status between IFNα- (A) and placebo-treated mice (B).

A B

Chapter 5

91

5.3.6. Interferon α Treatment Significantly Reduces Numbers of Activated

Hepatic Stellate Cells

Activated HSC stain positive for αSMA. These cells play a vital role in tissue remodeling

and fibrosis, and have been previously implicated in HPC-mediated liver repair.

Immunohistochemical staining with αSMA was performed in order to corroborate with

results obtained from fibrosis scoring and shed more light on the relationship between

activation of these myofibroblasts and the progenitor cell response. Treatment with IFNα

caused a mean reduction of almost 50% in the numbers of activated HSC (*p<0.05,

Figure 5.8A). It is important to take note of this finding despite the lack of apparent

change in fibrotic status.

IFNα Placebo0

10

20

30

40

*

%α

SMA

-pos

itive

cel

ls c

ells

Figure 5.8. Treatment with IFNα significantly reduced numbers of activated HSC. HSC which stained positive for αSMA were reduced by almost 50% in the IFNα-treated group (*p<0.05, A). Representative photographs of αSMA-positive cells taken from IFNα and placebo animals (B & C).

A

B

C

Chapter 5

92

5.3.7. Phosphorylation of STAT-3 as a Molecular Mechanism of Interferon α

Action

Thus far we have presented convincing data that IFNα causes a direct effect on the HPC

population in the chronically injured liver, and we proposed this to be a mechanism by

which interferon therapy reduces the risk of HCC in chronic hepatitis C patients.

However, it is also important to determine how IFNα governs the molecular events

which lead to changes such as inhibition of proliferation, induction of apoptosis and

differentiation, as well as changes in the cytokine profile. In order to address this issue,

livers from mice on the CDE diet were analyzed for STAT-3 phosphorylated at the

Ser705 residue using immunohistochemistry.

A significantly higher percentage of cells stained positive for phosho-STAT-3 in the

IFNα treated group compared to placebo. Mice treated with IFNα had a mean of 2.66%

phospho-STAT-3-positive cells as compared to 1.06% in the placebo (***p<0.001,

Figure 5.9). This proposes a molecular mechanism by which IFNα could exert its effects,

since STAT-3 is a major component of the Jak-STAT pathway and has been implicated

in modulating proliferation, apoptosis and differentiation 176,249,287,288.

Chapter 5

93

Figure 5.9. Treatment with IFNα significantly increased numbers of phospho-STAT-3-positive cells. Cells which stained positive for phospho-STAT-3 were increased by more than 2-fold in the IFNα treated group (***p<0.001, A). Representative photographs of phospho-STAT-3-positive cells taken from IFNα-treated and placebo animals (B & C).

A C

B

IFNα Placebo0

1

2

3 ***

% S

TAT-

3-P

-pos

itive

cel

ls

Chapter 5

94

5.4. Discussion

The results from these experiments support the findings presented in preceding chapters.

Firstly, we demonstrated that IFNα treatment dramatically reduced numbers of MPK-,

A6- and c-kit-positive HPC in mice fed the CDE diet. It is also important to note that a

reduction in percentage of c-kit-positive cells was also observed in Chapter 3 after the

chronic hepatitis C patients were treated with IFNα. In concurrence with results

presented in Chapter 4, we show in this current study that IFNα treatment markedly

reduces proliferation in vivo as demonstrated by the reduction of PCNA-positive HPC,

while apparently boosting proliferative status of the hepatocytes. Additionally, we show

that IFNα causes a slight increase in apoptotic HPC without affecting hepatocytes. This

demonstrates that the effects of IFNα on apoptosis and proliferation are somehow

differential between the two cell types.

Beyond the cellular level, IFNα also appears to modulate the levels of cytokines and

growth factors that have been previously implicated in recruitment, activation and

expansion of the HPC compartment 154. The reduction in hepatic levels of IFNγ in the

IFNα treated group correlates with lower numbers of HPC, since IFNγ has been

previously shown to be vital to proliferation of HPC 157,224. Similarly, reduced levels of

LTβ in the IFNα treated group was expected since levels of LTβ have been shown to be

tightly correlated to proliferation of HPC and thus postulated to be required for activation

and expansion of the compartment 154,156,157.

Chapter 5

95

No significant change in fibrotic status was observed, however, there were significantly

fewer activated HSC in the IFNα-treated animals. These cells are the principal fibrogenic

cell type of the liver and the reduced numbers could possibly be related to less fibrosis,

which may have been observed if the experimental time points extended further. This is

supported by the reduction in hepatic levels of TGFβ in the IFNα treated mice, a growth

factor whose primary function in liver fibrogenesis is to stimulate deposition of ECM.

Moreover, IFNα was previously suggested to reduce fibrosis in non-responding chronic

hepatitis C patients by antagonizing the TGFβ/Smad3 pathway 85. Results from this study

suggests that IFNα therapy improves patient prognosis by keeping the population of HPC

under control as well as antagonizing growth factors that contribute to fibrosis.

Recent work by Clouston and colleagues established a relationship between fibrosis seen

in chronic hepatitis C and numbers of HPC 289. They proposed that blockage in

proliferation of HCV-infected cells and steatotic hepatocytes caused the default activation

of the otherwise dormant HPC compartment. Taken together with the findings in this

study, it appears that the HPC compartment is activated by the inability of injured

hepatocytes to restore liver function, and the expansion of the compartment is driven

inflammatory cytokines. Interferon therapy plays a vital role in keeping this population

under control by driving differentiation and hence limiting the opportunities for the HPC

to become tumorigenic.

No significant changes in TNFα or IL-6 levels were observed in this present study, which

was surprising since both cytokines have been previously associated with increased

Chapter 5

96

progenitor cell numbers particularly with the CDE diet 154,155,168,282. Recently, our group

showed that IL-6 can induce LTβ expression by HPC 282, although findings from this

current study demonstrate that this may not always be true. However we can postulate

that either (i) IFNα treatment decreased LTβ production by other cells of the liver, or (ii)

IFNα treatment caused a selective reduction of LTβ levels in the liver without

significantly affecting IL-6 levels in a manner not currently understood.

Interferon treatment did not appear to change expression of TNFα, which is surprising as

this cytokine and its receptor has previously been shown to be essential for an adequate

HPC response 155. In this current study, IFNα appears to reduce the HPC response

without altering TNFα expression, suggesting that TNFα is but one of many cytokines

needed for orchestration of the HPC response.

In the final portion of this chapter, we set out to elucidate a molecular mechanism by

which IFNα exerts its effect on the HPC population. We tested its effects on

phosphorylation of STAT-3. The increased numbers of phospho-STAT-3-positive cells in

the IFNα treated mice illustrate a role played by the Jak-STAT pathway in the IFNα

induced effects on the HPC compartment in the CDE model of chronic liver injury. As

previously detailed in Chapter 1, IFNα is able to activate multiple IFN-responsive genes

using the Jak-STAT pathway. From the results obtained from the current study, it

suggests that IFNα uses this pathway to induce changes in proliferation, apoptosis and

differentiation, as well as altering the cytokine profile to control the HPC population in

the CDE model. Increased numbers of phospho-STAT-3 progenitor cells also indicate

Chapter 5

97

that the changes in proliferative status, apoptosis and differentiation seen in Chapter 4,

were a result of IFNα signaling through the Jak-STAT pathway. At this juncture it should

be acknowledged that IFNα may utilize other pathways such as the p38 MAPK and Erk

pathways, however the in-depth study of IFNα signaling was beyond the scope of this

study.

In conclusion, we confirmed that IFNα reduced HPC numbers both in vivo and in vitro.

Results from this study concur with data reported in Chapters 3 and 4. IFNα treatment of

mice placed on CDE diet reduces both MPK- and A6-positive HPC. In addition to its

effect on proliferation, apoptosis and differentiation as described in Chapter 4, we

showed that IFNα also impacts hepatic cytokines and growth factors that have been

previously implicated in the progenitor cell response. While levels of IFNγ and LTβ

decreased in a predictable manner in correlation to reduced HPC numbers with IFNα

treatment, IL-6 and TNFα defied the odds. This demonstrates that we know less about

this heterogeneous compartment of cells than claimed and the balance between

population expansion and control is far more complex than initially thought. Finally, we

confirmed use of phospho-STAT-3 by IFNα to incite molecular events resulting in

control of the HPC population in the CDE model, hence implicating a role for the Jak-

STAT pathway in the HPC response. This study only scrapes the surface of the

seemingly insurmountable task of uncovering all the molecular aspects of the actions of

IFNα in controlling the HPC response. Many questions remain concerning the various

members of the Jak-STAT pathway, their role in IFNα mediated control of the HPC

Chapter 5

98

compartment and the alternate pathways used by IFNα to reduce HPC numbers during

instances of chronic liver injury.

Chapter 6

99

6. Effects of Interferon γ on Hepatic Progenitor Cells in

vitro

6.1. Introduction

Liver regeneration mediated by HPC has become a widely accepted concept in recent

years. The extent of the HPC response seen during chronic liver injury varies with

severity of liver disease 69. Whilst regeneration is important for maintenance of liver

function, HPC proliferation may also pose risks for genesis of HCC. Therefore, it is

important to understand the role of cytokines and growth factors which control the HPC

response.

Understanding of the role of IFNγ, another potential regulator of HPC activation,

recruitment and expansion has evolved over the past year 157,224. Bisgaard and colleagues

used suppression subtractive hybridization to demonstrate the importance of IFNγ and

several genes modulated by IFNγ including the α-subunit of its receptor, gp91phox, IL-

1β, LFA-1, EIF-2 associated 67 kd protein and AFP 223. Lowes and colleagues suggested

that IFNγ may act synergistically with other growth factors to initiate proliferation 281.

Akhurst and colleagues later confirmed that IFNγ was essential for HPC response by

demonstrating that IFNγ -/- mice showed attenuated HPC response when placed on the

CDE diet 157. Brooling and colleagues showed that IFNγ could inhibit hepatocyte

proliferation while inducing proliferation of their HPC cell lines when used in

combination with TNFα or lipopolysaccharide (LPS) 224. These observations suggest that

Chapter 6

100

IFNγ and other cytokines invoked during toxic and inflammatory reactions may

contribute to the type of cells which repopulate the injured liver.

Briefly, the aims of this chapter were to:

(i) elucidate effects of IFNγ on proliferation, apoptosis and differentiation of the

HPC cell lines, PIL-2 and PIL-4.

(ii) verify results using primary HPC derived from the CDE mouse model, since

previous studies have shown varying effects of IFNγ on HPC depending on cell lines

and when used in combination with other cytokines and growth factors.

Chapter 6

101

6.2. Methods

6.2.1. Detecting the Presence of α and β Chains of the Interferon γ Receptor

The presence of α and β chains of the IFN receptor was determined by RT-PCR. The

following primer sequences were used: IFNγ receptor α chain (forward) 5’-AGA TCC

TAC ATA CGA AAC ATA CGG-3’; IFNγ receptor α chain (reverse) 5’-TTT CTG TCA

TCA TGG AAA GGA GGG ATA CAG-3’; IFNγ receptor β chain (forward) 5’-TCG

TTT TCC CAG CTT GCG GCC-3’; and IFNγ receptor β chain (reverse) 5’-AAG CTA

TAT TCC ACC TGG TA-3’. Each RT-PCR reaction mixture contained 1X reaction

buffer (Fisher Biotech, Perth, Australia), 1.25mM MgCL2 (Fisher Biotech), 0.25mM

dNTPs (Fisher Biotech), 2 units of Platinum Taq (Invitrogen) and 2µL of cDNA

synthesized from DNAse-treated RNA as according to protocols detailed in Sections

2.3.3 to 2.3.4. The cycling conditions used were as follows: 95oC 5 minutes denaturation,

95oC 20 seconds, 55oC 20 seconds and 72oC 30 seconds for 30 cycles, and extension at

72oC for 5 minutes. The products for the α and β chains were 187 bp and 131 bp

respectively.

Chapter 6

102

6.2.2. Measuring of the Effect of IFNγ on Proliferation of Hepatic Progenitor

Cells Using MTT Assay and BrdU Incorporation

PIL-2 and PIL-4 cells were grown and serum starved in Williams E media as previously

described in Section 2.2.1 and 2.2.2 respectively and stimulated with 0.025 - 50ng/mL

recombinant murine IFNγ (R&D Systems, Minneapolis, MN) for 24 hours. Change in

mitochondrial activity was measuring using MTT assay as described in Section 4.2.2.

Changes in mitochondrial activity were confirmed in primary HPC using a BrdU

incorporation assay (Calbiochem, San Diego, CA, USA) according to manufacturer’s

instructions. BrdU incorporation is a well-established alternative to [3H] thymidine

incorporation. BrdU is incorporated into newly synthesized strands of DNA of actively

proliferating cells. The double stranded DNA is partially denatured and BrdU is

immunochemically detected to allow assessment of the population of cells which are

actively synthesizing DNA.

Briefly, 1, 000 cells were seeded into each well of a 96 well plate. These cells were

allowed to adhere in media containing 10% FCS and then serum starved overnight in

media without FCS. They were stimulated with IFNγ for 24 hours with media containing

20ng/mL IFNγ. Working stock of BrdU was made by diluting BrdU label 1:2000 with

fresh culture media, 20µL of BrdU working solution was added to each well and

incubated for 24 hours at 37oC. The contents of the wells were then removed and 200µL

of Fixtative/Denaturing Solution was added to each well and incubated for 30 minutes at

room temperature. The contents of the wells were then removed and BrdU incorporation

Chapter 6

103

was detected by added 100µL of Anti-BrdU antibody diluted 1:100 with Antibody

Dilution Buffer to each well and incubated for 1 hour at room temperature. The wells

were washed 3 times with 1X Wash Buffer and 100µL of peroxidase goat anti-mouse

IgG HRP conjugate in Conjugate Diluent was added to each well and incubated for 30

minutes at room temperature. The wells were washed 3 times with 1X Wash Buffer and

then rinsed with distilled water. 100µL of Substrate Solution was added to each well and

incubated for 15 minutes at room temperature in the dark to allow the development of a

coloured product that could be quantified using a spectrophotometer. The reaction was

stopped by adding 100µL of Stop Solution to each well. Absorbance was read at dual

wavelengths of 450 – 540nm within 30 minutes of stopping the reaction.

6.2.3. Measuring the Effect of IFNγ on Apoptosis Using TUNEL Assay

PIL-2 and PIL-4 cells were grown in 4-well chambered slides and stimulated with

20ng/mL IFNγ for 24 hours and the effect on apoptosis was determined using the

TUNEL assay (Promega) as described in Section 2.10. The number of TUNEL-positive

cells were counted at 400X magnification and expressed as a percentage of total numbers

of cells in the field.

Chapter 6

104

6.2.4. Determining the Effect of Interferon γ on Differentiation Status of the

Hepatic Progenitor Cells

PIL-2 and PIL-4 cells were grown in media containing 20ng/mL of IFNγ for 8 days.

Expression of differentiation markers was determined using qPCR as detailed in Section

4.2.4.

6.2.5. Primary Hepatic Progenitor Cell Isolation

BrdU incorporation involving primary HPC were undertaken to confirm results obtained

using the PIL cell lines. C57Bl/6 mice were placed on a CDE diet as described in Section

2.5.1. The portal vein was cannulated for perfusion with EGTA perfusion buffer (0.4mM

EGTA, 136mM NaCl, 4.7 KCl, 1.2mM KH2PO4, 0.65mM MgSO4 X 7H20, 10mM

HEPES, pH 7.4) at 5mL/min for 10 mins. This was followed by perfusion with

collagenase perfusion buffer (66.7mM NaCl, 6.7mM KCl, 4.8mM CaCl2 X 2H20) for 11

minutes. The livers were collected in sterile petri dishes and cells were dissociated from

the liver capsule using sterile forceps and scissors while immersed in wash media

(Williams E media, 5%v/v FCS, 1%v/v Gentamicin). The cell suspension was transferred

into a 50mL tube and centrifuged at 300 x g for 8 mins. The supernatant was removed

and the cell pellet was resuspended in 12.5mL digestion solution [1X PBS, 20mg/mL

Pronase (Sigma), 0.2% DNase I (Sigma), 1.11X10-4 units of Type VIII collagenase

(Sigma)] and 1mL Trypsin:EDTA (Thermoelectron Corporation, Waltham,

Massachusetts, USA) and incubated at 37oC with rocking.

Chapter 6

105

Following digestion, the cell suspension was filtered through two layers of sterile gauze

into a fresh 50mL tube and centrifuged at 300 x g for 8 mins. The pellet was resuspended

in wash medium and centrifuged again. The supernatant was removed and resuspended in

10mL of wash medium. The suspension was underlaid carefully with 20% Percoll

(Amersham Biosciences, UK) followed by 50% Percoll and centrifuged at 1400 xg for 20

mins at 4oC. Cells separated at the interface between media and 20% Percoll fraction

were transferred into a fresh tube containing an equal volume of wash medium. These

cells were centrifuged at 300 x g for 8 minutes to remove Percoll before cell counting and

plating. Cells were plated at a density of 1-2 X 106 cells/mL in 6 or 24 well plates. This

method has been documented to yield HPC of approximately 90% purity.

Chapter 6

106

6.3. Results

6.3.1. Both α and β Chains of IFNγ Receptor are Expressed by PIL-2 and

PIL-4

The murine HPC cell lines used in this study expressed both chains of the IFNγ receptor,

as illustrated in Figure 6.1, suggesting that they are responsive to this cytokine.

Figure 6.1. Both chains of the IFNγ receptor are expressed by PIL-2 and PIL-4 cell lines. The α chain of the IFN receptor is expressed by PIL-2 and PIL-4 as seen in lanes 1 and 2, respectively. The β chain of the IFN receptor is expressed by PIL-2 and PIL-4 as seen in lanes 3 and 4, respectively.

200bp

α - subunit β - subunit

1 2 3 1 2 3 4 5 6

Chapter 6

107

6.3.2. Interferon γ Inhibits Proliferation of PIL-2 and PIL-4 in vitro

Treatment of PIL-2 and PIL-4 cells with IFNγ caused an inhibition of mitochondrial

activity in a dose-dependent manner as illustrated by MTT assay (Figure 6.2). The LD50

was 20ng/ml for PIL-2 and 5ng/mL for PIL-4 cells, respectively.

0.25 0.5 1 2 4 8 16 32 64 1280.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

IFNγ (ng/mL)Mito

chon

dria

l act

ivity

nor

mal

ised

aga

inst

neg

ativ

e co

ntro

l

Figure 6.2. Interferon γ reduced mitochondrial activity. Following 24 hours culture in media containing a spectrum of concentrations of IFNγ, mitochondrial activity is reduced in both PIL-2 (A) and PIL-4 (B) cell lines. LD50 was 20ng/mL for PIL-2 cells and 5ng/mL for PIL-4 cells.

A

B

0.125 0.25 0.5 1 2 4 8 16 32 64 1280.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

IFNγ (ng/mL)

Mito

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l act

ivity

nor

mal

ised

aga

inst

con

trol

Chapter 6

108

6.3.3. Interferon γ Induced Apoptosis in PIL-2 But Not PIL-4

Treatment of PIL-2 and PIL-4 with IFNγ increased apoptosis. Numbers of apoptotic cells

were significantly higher in the IFNγ treated PIL-2 cells compared to untreated cells, with

more than a 3-fold increase in the proportion of apoptotic cells from a mean of 0.185% in

the untreated cells to a mean of 0.753% in the IFNγ treated cells (Figure 6.3A *p<0.05).

In the PIL-4 cell line, the percentage of apoptotic cells did not increase significantly with

IFNγ treatment (Figure 6.3B).

IFN γ untreated0.0

0.2

0.4

0.6

0.8

1.0 *

% T

UN

EL-p

ositi

ve c

ells

IFNγ untreated0.0

0.5

1.0

1.5

2.0

2.5%

TU

NEL

-pos

itive

cel

ls

Figure 6.3. Treatment of HPC with interferon γ increased numbers of apoptotic cells. The percentage of apoptotic PIL-2 cells increased significantly (A, *p<0.05) whilst there was no substantial change in the number of apoptotic PIL-4 cells (B).

A B

Chapter 6

109

6.3.4. Treatment of PIL-2 and PIL-4 with Interferon γ Changed Expression of

Hepatic Maturation Markers

Treatment of PIL-2 or PIL-4 cells with 20ng/mL IFNγ did not result in changes in

albumin gene expression (Figure 6.4A & B). However, it did cause significant changes in

expression of other markers in the two cell lines. IFNγ treatment resulted in ~43-fold

reduction in CK19 expression in PIL-2 cells and 100-fold increase in PIL-4 cells (Figure

6.4C, *p<0.05 & D, *p<0.05). In addition to these changes, IFNγ treatment resulted in a

significant 15-fold reduction in AFP expression in PIL-2 cells (Figure 6.4E, *p<0.05)

while PIL-4 cells increased their expression of AFP by 40% (Figure 6.4F, ***p<0.001).

Expression of G6Pase reduced by 95% in PIL-2 cells following IFNγ treatment (Figure

6.4G, *p<0.05), but doubled in PIL-4 cells (Figure 6.4H, *p<0.05).

Chapter 6

110

A

Figure 6.4. Interferon γ treatment affects expression of maturation markers by PIL cells. Following 8 days of culture in media containing 20ng/mL IFNγ, there was no change in albumin expression in PIL-2 or PIL-4 cells (A & B). Expression of CK19 decreased approximately 43-fold in PIL-2 cells (C, *p<0.05) and increased 100-fold in PIL-4 cells (D, *p<0.05). Note: All mRNA levels of hepatic maturation markers were normalized against mRNA levels of β-actin.

IFNγ Untreated0.0

0.2

0.4

0.6

0.8

1.0

1.2A

lbum

in m

RN

A le

vels

IFNγ Untreated0.0

0.2

0.4

0.6

0.8

1.0

1.2

Αlb

umin

mR

NA

leve

ls

B

IFNγ Untreated0.00

0.01

0.02

0.03

*CK

19 m

RN

A le

vels

C IFNγ Untreated

0.0000

0.0025

0.0050

0.0075

0.0100 *C

K19

mR

NA

leve

ls

D

Chapter 6

111

Figure 6.4. Interferon γ treatment affects expression of maturation markers by PIL cells (continued). Following 8 days of culture in media containing 20ng/mL IFNγ, PIL-2 expression of AFP was reduced by 15-fold (E, *p<0.05) but increased by almost 40% in PIL-4 cells (F, ***p<0.001). Additionally, a 95% reduction in G6Pase expression in PIL-2 cells (G, *p<0.05) was accompanied by a 2-fold increase in expression by PIL-4 cells (H, *p<0.05). Note: All mRNA levels of hepatic maturation markers were normalized against mRNA levels of β-actin.

IFNγ untreated0.0000

0.0001

0.0002

0.0003

*G6P

ase

mR

NA

leve

ls

IFNγ Untreated0

500

1000

1500

2000

*AFP

mR

NA

leve

ls

E IFNγ Untreated0

50

100

150

200

250

300

350***

AFP

mR

NA

leve

ls

F

G IFNγ Untreated0

2e-006

4e-006

6e-006*

G6P

ase

mR

NA

leve

ls

H

Chapter 6

112

6.3.5. Confirmation of Cell Line Data in Primary Progenitor Cells Significant growth inhibition was observed in primary HPC following 72 hours culture in

media supplemented with 20ng/mL IFNγ as shown using BrdU incorporation assay

(Figure 6.5).

Vehicle 2ng/mL 20ng/mL0

50

100

150

200

**

IFNγNum

ber o

f Brd

U-p

ositi

ve c

ells

per

wel

l

Figure 6.5. Interferon γ is anti-proliferative to primary hepatic progenitor cells in culture. Primary HPC isolated from the livers of mice fed a CDE diet showed significantly reduced growth following treatment with 20ng/mL IFNγ for 72 hours (**p<0.01).

Chapter 6

113

6.4. Discussion

IFNγ is a cytokine implicated in numerous molecular events within the cell. It has been

shown to have immunoregulatory properties, and has also been implicated in the control

of cell cycling and apoptosis. IFNγ has been previously shown to increase sensitivity of

tumor cell lines to TNFα and anti-Fas antibody-mediated cell death via a p53-

independent apoptotic pathway 290. Mediation of IFNγ apoptosis by IRF-1 via caspase-1

expression has also been documented 291. The current work extends our understanding of

the effect of IFNγ on p53-null and primary HPC by demonstrating that IFNγ is pro-

apoptotic and anti-proliferative to HPC in vitro.

Treatment of HPC with IFNγ inhibited proliferation in all cell lines, accompanied by an

increase in apoptosis which was most pronounced in PIL-2 cells. Additionally, IFNγ

caused significant changes in the expression of AFP, CK19 and G6Pase but not albumin.

These changes in expression of differentiation markers were seen in both PIL-2 and PIL-

4 cell lines. Unlike the results reported in Chapter 4, there was no clear pattern indicative

of differentiation in either cell line. However it is worthwhile noting that the pattern of

expression of maturation markers differ between the two cell lines. Although albumin

mRNA levels in both cell lines remained unchanged after IFNγ treatment, the cytokine

caused opposing changes in the expression of other hepatic maturation markers. IFNγ

treatment caused a decrease in the expression of CK19, AFP and G6Pase in the PIL-2

cells, but an increase in the expression of the same markers in the PIL-4 cells. While we

were unable to show that IFNγ can stimulate differentiation of the HPC in culture, this

Chapter 6

114

does illustrate one of many differences between these two cell lines. They have also been

shown in this chapter to vary in their susceptibility to IFNγ-induced apoptosis.

Additionally the two cell lines have been shown to differ in terms of their ability to form

tumours in vivo 93.

The findings presented in this chapter contrast with findings recently published by

Brooling and colleagues who reported that the cytokine was pro-proliferative using their

liver progenitor cell lines 224. While the reasons for this discrepancy remain unclear, it is

worthwhile noting that IFNγ was pro-proliferative in the LE-6 cells but anti-proliferative

in the LE-2 cells. In the latter cell line, IFNγ diminished the pro-proliferative effect of

TNFα when the two cytokines were used in combination, thus illustrating that perhaps

IFNγ is not directly pro-proliferative on all HPC. Some explanation for differences

between the studies may lie within the origin of the cells. The PIL cells were derived

from mouse oval cells, while LE cells were derived from rat oval cells. In addition, the

two cell lines are cultured in different conditions; the PIL cells are cultured under serum-

free conditions following overnight subculture in Williams E media containing 10% fetal

bovine serum to allow for adherence, while the LE cells are continuously grown in

Dulbecco’s modified Eagle’s media containing at least 1% serum. The latter conditions

allow for unknown co-factors to interact and possibly interfere with the effect that IFNγ

has on apoptosis and proliferation of the cells, thus potentially accounting for some of the

contrasting effects that were observed between the cell lines. More importantly, PIL cells

are p53-null while LE cells have an intact p53 tumor suppressor gene. The p53 gene is

located on the short arm of chromosome 17 and causes cell cycle arrest during instances

Chapter 6

115

of DNA damage, via p21, which has been documented to play a vital but complex role in

IFNγ-mediated growth inhibition 292. p53 has long been recognised for its function as a

cell cycle checkpoint protein through the transactivation of genes which encode proteins

with growth suppressing activities exerting its effect during the G1 phase of the cell

cycle. Since the LE cell lines used by Brooling and colleagues have an intact p53, while

the PIL cells are p53 null, this strongly indicates that the IFNγ-induced apoptosis and

inhibition of proliferation seen in PIL cells were p53-independent events and the cytokine

may have caused differential effects on the PIL and LE cells due to the presence/absence

of this major cell cycle checkpoint protein. However, it was interesting that growth

inhibition and apoptosis were also seen in the primary oval cells and it is likely that IFNγ

exerted its effects on the primary oval cells in a p53-independent manner as well.

In conclusion, IFNγ exerts an anti-proliferative and pro-apoptotic effect on PIL cells as

well as primary oval cells. This effect was dose-dependent. IFNγ induced differentiation

in the PIL-4 cells but appeared to have little effect on differentiation of the PIL-2 cells,

and this is likely attributed to differing susceptibilities of the two PIL cell lines to the

effects of IFNγ.

Chapter 7

116

7. Effects of Interferon γ on Hepatic Progenitor

Cells in vivo

7.1. Introduction

In 1999, Bisgaard and colleagues suggested that IFNγ plays a central role in mediating

differential regenerative responses invoked during HPC/hepatocyte-mediated liver

regeneration 223. More recently, Sun and Gao have documented negative regulation of

hepatocyte-mediated liver regeneration by IFNγ produced by natural killer cells suggesting

that IFNγ is anti-proliferative to hepatocytes 293. Brooling and colleagues confirmed this in

their recent paper, demonstrating that IFNγ is anti-proliferative to AML12 hepatocytes but

pro-proliferative to their LE-6 HPC cell line 224. Our group has also recently published

findings confirming the requirement for IFNγ in mediating the HPC response to the CDE

diet 157. These studies show that IFNγ could play a pivotal role in liver regeneration

following chronic liver injury through the concurrent inhibition of hepatocyte proliferation

and growth-stimulatory effects on the HPC population. Somewhat surprisingly, results

from Chapter 6 suggest that IFNγ may be inhibitory to HPC in vitro – providing data which

seemingly contradicts what would be expected on the basis of published in vivo work.

Thus, the aim of this chapter was to clarify these discrepancies and examine the direct

effects of IFNγ on the HPC population during liver injury in vivo.

Chapter 7

117

7.2. Methods

7.2.1. Treating CDE-fed mice with Interferon γ

To determine the in vivo effects of IFNγ on the HPC population, four-week old C57Bl/6

male mice weighing between 14 to 16g were placed on the CDE diet to induce proliferation

of HPC. Animals were administered either 2ng of recombinant murine IFNγ (R&D

Systems) or saline (placebo) intraperitoneally once a day. Mice were sacrificed two weeks

later. Livers were perfused with saline, and portions of liver were sampled for histological

studies. All animal experiments were performed in a pathogen-free animal holding facility

in accordance with guidelines of the National Health and Medical Research Council of

Australia and approved by the University of Western Australia Animal Ethics Committee.

7.2.2. Feeding the CDE Diet

Mice were fed the CDE diet as described in Section 5.2.2.

Chapter 7

118

7.2.3. Immunohistochemical Staining for A6, M2-Pyruvate Kinase (MPK) and

Stem Cell Factor Receptor (c-kit)

Changes in the numbers of hepatic progenitor cells brought about by IFNγ treatment were

studied using immunohistochemical staining for A6, MPK and c-kit as detailed in Section

5.2.3.

7.2.4. Sirius Red Staining for Detecting Change in Fibrosis

Tissue sections were stained with sirius red and scored for extent of fibrosis as described in

Section 5.2.8.

7.2.5. Determining Change in Apoptotic Status Using TUNEL Staining

The changes in apoptotic status as brought about by IFNγ treatment was determined using

TUNEL staining as detailed in Section 2.10.

Chapter 7

119

7.2.6. Determining Changes in Proliferative Status Using Cytokeratin/Ki67

Double Immunohistochemical Staining

For analysis of in situ proliferation of the HPC population, double immunohistochemical

staining for Ki67 and cytokeratin was performed. Paraffin sections were cut from formalin-

fixed tissues and deparaffinised. Antigen retrieval involved boiling sections in EDTA

buffer (0.1M, pH 8.0) for 20 minutes. Following this, sections were allowed to cool for at

least 20 minutes before washing the slides in TBS and blocking against endogenous

peroxidases with 3% H2O2 for 10 minutes. The sections were then blocked against non-

specific antibody binding by blocking with a serum-free protein block (Dako) for 15

minutes. Anti-cytokeratin (Dako) was diluted 1:400 and applied onto tissue sections

overnight at room temperature. Detection was performed the next day using a horseradish

peroxidase coupled secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA,

USA) and visualized using aminoethycarbazole solution (Sigma). Sections were re-blocked

in 3% H2O2 and incubated in rat anti-mouse Ki67 (Dako) diluted 1:10 for 1 hour at 37oC.

Detection was performed using anti-rat IgG coupled to horseradish peroxidase and

visualized using chloronaphthol solution (Pierce, Rockford, IL, USA). The slides were

mounted in Kaiser’s glycerol mountant. Cells in non-overlapping periportal regions were

counted at 400X magnification and expressed as a percentage of hepatocytes in the field.

Cytokeratin-positive cells stained red in the cytoplasm while Ki67-positive cells stained

blue in the nucleus. Only cells with blue nuclear staining and red cytoplasmic staining were

considered as proliferating HPC, and thus included in the cell counts.

Chapter 7

120

7.2.7. Detection of Inflammatory Influx Using CD45 Immunohistochemical

Staining

Influx of inflammatory cells was detected using CD45 immunohistochemical staining.

Briefly, deparaffinised tissue sections were incubated with 20µg/mL proteinase K solution

in a humidified chamber for 20 minutes at 37oC. Sections were then rinsed twice in TBS

and blocked for endogenous peroxidases using 3% H202 for 10 minutes. Sections were also

blocked against endogenous biotin using a biotin blocking kit (Dako) before incubation

with serum-free blocking solution for 15 minutes at room temperature. Sections were then

washed and exposed to rat anti-mouse CD45 diluted 1:20 (BD Pharmingen, Bedford, MA,

USA) for 1 hour at room temperature. Sections were then rinsed thoroughly in TBS and

exposed to secondary biotinylated rabbit anti-rat IgG for 30 minutes at room temperature.

Sections were then washed in TBS and visualized using HRP-Streptavidin and liquid DAB.

Sections were dehydrated and cleared prior to mounting.

7.2.8. Analysis of Hepatic Cytokine Profile

Changes in hepatic expression of cytokines were determined using qPCR as described in

Section 5.2.7.

Chapter 7

121

7.3. Results

7.3.1. Interferon γ Increased Numbers of MPK-, A6- and c-kit-positive Hepatic

Progenitor Cells in CDE-fed Mice

Treatment with IFNγ significantly increased the numbers of HPC in the CDE mouse model.

A 3-fold increase in the percentage of A6-positive cells was observed in the IFNγ treated

CDE-fed mice compared to placebo (Figure 7.1, **p<0.005).

Figure 7.1. Interferon γ treatment increased numbers of A6-positive cells in mice placed on CDE diet. A6-positive cells increased by approximately 3-fold in IFNγ treated group compared to placebo (**p<0.005, A). Representative photographs taken of A6-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 200X magnification.

A IFN γ Placebo

0.0

2.5

5.0

7.5

10.0 **

% A

6-po

sitiv

e ce

lls

C

B

Chapter 7

122

Treatment with IFNγ also caused a significant increase in the percentage of MPK-positive

cells. The proportion of MPK-positive cells increased from a mean of 6 % in the placebo

animals to a mean of 20 % illustrating a greater than 3-fold increase (Figure 7.2,

**p<0.005).

Figure 7.2. Interferon γ treatment increased numbers of MPK-positive cells in mice placed on CDE diet. MPK-positive cells increased by more than 3-fold in IFNγ treated group compared to placebo (**p<0.005, A). Representative photographs taken of MPK-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 400X magnification.

IFN γ Placebo0

5

10

15

20

25 **

% M

PK-p

ositi

ve c

ells

A

C

B

Chapter 7

123

IFNγ treatment also caused an increase in the percentage of c-kit-positive cells in the CDE-

fed mice. The c-kit-positive cells increased from a mean of 0.5 % in placebo animals, to a

mean of 2.1 % in IFNγ-treated animals (Figure 7.3, ***p<0.001).

Figure 7.3. Interferon γ treatment increased numbers of c-kit-positive cells in mice placed on CDE diet. The proportion of c-kit-positive cells increased by more than 3-fold in IFNγ−treated group compared to placebo (***p<0.001, A). Representative photographs taken of c-kit-positive progenitor cells in IFNγ−treated (B) and placebo animals (C) at 400X magnification.

IFN γ Placebo0.0

0.5

1.0

1.5

2.0

2.5 ***

% c

-kit-

posi

tive

cells

A

C

B

Chapter 7

124

7.3.2. Interferon γ Exerted a Differential Effect on Apoptosis of Hepatic

Progenitor Cells and Hepatocytes in vivo

Treatment of the CDE-fed mice with 2ng of IFNγ per day did not affect the proportion of

apoptotic progenitor cells (Figure 7.4A), however it resulted in a 10-fold increase in the

percentage of TUNEL-positive hepatocytes (Figure 7.4B, **p<0.01).

Chapter 7

125

IFNγ Placebo

0.0

0.1

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Figure 7.4. Treatment with IFNγ increases proportion of apoptotic hepatocytes but not hepatic progenitor cells. The proportion of TUNEL-positive HPC was not significantly affected by IFNγ treatment (A). However, it did significantly increase the percentage of TUNEL-positive hepatocytes by approximately 10-fold (B, **p<0.01). Representative photographs of TUNEL-staining in IFNγ (C) and placebo (D) samples at 400X magnification.

A B

C

D

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7.3.3. Interferon γ Exacerbated Inflammation and Fibrosis in CDE-fed Mice

Greater amounts of inflammation and fibrosis were detected in the mice administered IFNγ

compared to placebo. Treatment of CDE-fed mice with IFNγ caused an average 4-fold

increase in numbers of inflammatory cells compared to placebo animals as shown by CD45

staining (Figure 7.5)

Figure 7.5. Treatment with IFNγ increased mean numbers of inflammatory cells as shown by CD45 immunohistochemical staining. Treatment of CDE-fed mice with IFNγ increased CD45-positive cells by 4-fold (A, **p<0.01). Representative photographs of CD45 staining in IFNγ-treated (B) and placebo animals (C) at 400X magnification.

A

C

B

Placebo IFNγ0.0

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In addition, greater morbidity and mortality were seen in the IFNγ group (2 out of 6

animals) compared to the placebo group (0 out of 6 animals). The median fibrosis score for

CDE-fed mice given placebo was 1.2, while the median fibrosis score for CDE-fed mice

given 2ng/mL IFNγ daily, was 2.3 as detailed below in Table 7.1. Representative

photographs of Sirius Red staining are shown below (Figure 7.6).

Table 7.1. Fibrosis staging of mice placed on CDE diet.

IFNγ Placebo

Stage 1 0 3

Stage 2 2 3

Stage 3 2 0

Stage 4 0 0

*Stage 1 – Mild fibrosis isolated to periportal regions *Stage 2 – Moderate fibrosis isolated to periportal regions *Stage 3 – Septal and bridging fibrosis between portal tracts *Stage 4 – Cirrhotic bands linking portal tracts

Figure 7.6. Representative photographs of sirius red staining collagen fibres in periportal regions of livers from CDE-fed mice administered 2ng/mL IFNγ daily (A) and vehicle (B) at 200X magnification.

A B

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7.3.4. Interferon γ Induced Changes in the Hepatic Cytokine Profile

Following 14 days of intraperitoneal administration of 2ng/mL IFNγ, changes were

observed in the hepatic levels of cytokines previously suggested to play key roles in

modulating the HPC response. Expression of TNFα doubled following IFNγ treatment

(Figure 7.7A, *p<0.05), while IL-6 expression increased approximately 46-fold (Figure

7.7B, *p<0.05). LTβ expression decreased 17-fold (Figure 7.7C, *p<0.05) while IFNγ

expression decreased by 1014-fold (Figure 7.7D, *p<0.05), to near negligible levels. Mean

gene expression of TGFβ was reduced by 35-fold in the IFNγ-treated mice compared to

placebo (Figure 7.7E, *p<0.05).

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IFNγ Placebo0.0

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leve

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Figure 7.7. Changes in hepatic cytokine profile following daily administration of 2ng/mL IFNγ to CDE-fed mice. mRNA levels of TNFα were doubled by IFNγ treatment (A), while expression of IL-6 increased by 46-fold (B). Concurrently, IFNγ treatment caused significant decreases in the hepatic expression of LTβ (C), IFNγ (D) and TGFβ (E). Note: mRNA levels of cytokines were normalized against mRNA levels of β-actin.

A

B

C

D

E

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7.3.5. Interferon γ Invoked Inflammatory Cell Infiltration and Exacerbated

Hepatocyte Damage During Early Stages of CDE-feeding

In order to determine the mechanisms which underlie the increased HPC response and

fibrosis observed in IFNγ-treated mice after 2 weeks of CDE feeding, a more detailed study

of earlier time points at 3, 5 and 7 days was undertaken as shown in Table 7.2 with 3 mice

in each group.

Table 7.2. Additional experimental time points and regimes

Time points Regime (treatment/diet)

Day 3 placebo/control IFNγ/control placebo/CDE IFNγ/CDE



Increased liver damage was seen in the IFNγ/CDE group, but no parenchymal damage was

observed in the IFNγ/control group, as reflected by ALT levels shown in Figure 7.8. Levels

of ALT were 9-fold greater in the IFNγ/ CDE group (1439 ± 387 U/mL) at day 3 compared

to the placebo/CDE group (164 ± 51 U/mL) at the same time point. However, ALT levels

decreased in the following days where it was 3.4-fold higher (971 ± 30 U/mL c.f. 282 ± 68

U/mL), and by day 7 there was no significant difference between the ALT levels of the two

groups. Notably, IFNγ did not cause liver injury without the background of the CDE diet.

All control animals fed the control diet had normal serum ALT levels (20-30 U/mL).

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Figure 7.8. Mean serum ALT measurements taken from mice administered vehicle (placebo) and 2ng/mL IFNγ daily with CDE or control diet, after 3, 5 and 7 days. Mice placed on control diets did not experience a change in ALT levels even when administered daily intraperitoneal injections of IFNγ. Elevation of ALT levels is expected in the CDE-fed mice, however, administration of IFNγ causes an exaggeration of initial liver damage at day 3, which eventually subsides to basal levels by day 7.

placeb

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The elevated ALT levels in animals fed the CDE diet is strongly indicative of liver injury;

an insult which is exacerbated by IFNγ treatment. In addition to this, infiltration by

inflammatory cells was observed in the periportal regions in IFNγ/control animals at Day 3

(Figure 7.9A) and to a lesser extent at Day 5 (Figure 7.9B). The clusters of inflammatory

cells ranged between ~20 to ~100 cells (indicated by arrows). By day 7, the phenotype was

no longer observed (Figure 7.9C). Influx of inflammatory cells was not detected in

placebo/control animals (Figure 7.9 D-F). In contrast, influx of inflammatory cells was

observed in IFNγ/CDE animals (Figure 7.9 G-I) but not placebo/CDE animals (Figure 7.9

J-L), with the former showing exacerbated inflammatory response accompanied by

parenchymal damage. This finding suggests that increased fibrosis and HPC response may

have been ‘primed’ by IFNγ administration which induced influx of inflammatory cells.

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A D

B E

C F

Figure 7.9. Treatment with IFNγ induces influx of inflammatory cells. Representative photographs of haematoxylin and eosin-stained tissue sections at 200X magnification. Pockets of inflammatory cells were observed in IFNγ/control animals at Day 3 (A) and to a lesser extent at Day 5 (B). This is no longer observed at Day 7 (C). Normal healthy liver architecture was noted in the placebo/control animals at Day 3, 5 and 7 (D-F respectively).

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G J

H K

I L

Figure 7.9 (continued). Treatment with IFNγ induces influx of inflammatory cells. There was a greater extent of liver damage in the IFNγ/CDE mice at Day 3 (G), 5 (H) and 7 (I) compared to the placebo/CDE mice at the same time points (J-K respectively). Pockets of inflammatory cells (indicated by arrows) were observed in the IFNγ/CDE but not in the placebo/CDE group.

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There is a basal level of cell turnover of hepatocytes seen in the two control groups -

placebo/control and IFNγ/control. Daily intraperitoneal administration of IFNγ does not

result in any significant effects on hepatocyte apoptosis when animals are placed on control

diets. The damaging effects of IFNγ on hepatocytes is evidenced by the massive increase in

apoptotic-hepatocytes in the IFNγ/CDE group as compared to the other groups (Figure

7.10). While the CDE diet does cause some degree of hepatocyte cell death, the mean

extent of hepatocyte apoptosis is 4-fold greater in the IFNγ/CDE group at day 3 compared

to placebo/CDE group at the same timepoint (*p<0.05). By day 5, there is no significant

difference in hepatocyte cell death between the two groups. IFNγ caused an initial rise in

hepatocyte death to 2.6%, which then decreased to 0.11% by day 7 – a 99% reduction in

hepatocyte apoptosis. The proportion of apoptotic hepatocytes seen at day 7 in the

IFNγ/CDE group was not significantly different to the basal hepatocyte turnover seen in the

placebo/control group at day 3, 5 or 7 (green).

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Figure 7.10 Treatment of CDE-fed mice with IFNγ increased hepatocyte apoptosis. Basal level of hepatocyte turnover is seen in mice on control diets. IFNγ treatment alone did not significantly increase the extent of apoptosis. Mice fed the CDE diet had increased hepatocyte death within the first 5 days of feeding. IFNγ treatment of CDE-fed mice caused an approximate 4-fold increase in the first 3 days compared to those given placebo (*p<0.05).

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Double immunohistochemical staining for Ki67 and cytokeratin was used to identify

proliferating HPC. Increased numbers of proliferating HPC were seen in the IFNγ/CDE

group as indicated by black bars in Figure 7.11. IFNγ treatment increased proliferation of

HPC even without the background of the CDE diet. The proportion of proliferating HPC

increased by 6-fold in mice given the control diet with IFNγ treatment compared to placebo

at day 3 (*p<0.05). Additionally, the proportion of proliferating HPC was 3 times in the

CDE/IFNγ group compared to CDE/Placebo group at day 3 (**p<0.01). Representative

photographs of Ki67/cytokeratin staining at 200X magnification are shown in Figure 7.12.

More detailed photographs of double immunohistochemical staining for Ki67/cytokeratin at

400X magnification are shown in Figure 7.13.

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Figure 7.11. Double immunohistochemical staining for Ki67 and cytokeratin identifies the population of actively proliferating HPC population. Treatment with IFNγ significantly increases proliferation of progenitor cells both control- (*p<0.05) and CDE- (**0<0.01) fed mice.

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Figure 7.12. Representative photographs (200X maginification) of double immunohistochemical staining with Ki67 and cytokeratin for identification of proliferating hepatic progenitor cells. Cytokeratin-positive cells stain red while Ki67 cells stain blue. Control/Placebo – Day 3 (A), Day 5 (B), Day 7 (C); Control/IFNγ – Day 3 (D), Day 5 (E), Day 7 (F).

A

B

C

D

E

F

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Figure 7.12 (continued). Representative photographs (200X maginification) of double immunohistochemical staining with Ki67 and cytokeratin for identification of proliferating hepatic progenitor cells. Cytokeratin-positive cells stain red while Ki67 cells stain blue. CDE/Placebo – Day 3 (G), Day 5 (H), Day 7 (I); CDE/IFNγ – Day 3 (J), Day 5 (K), Day 7 (L).

G

H

I

J

K

L

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Figure 7.13. Representative photographs of double immunohistochemical staining of Ki67 and cytokeratin shown at 400X magnification. Above photographs show double staining of CDE/Placebo (A) and CDE/IFNγ (B) at day 3. Only cells positive for both markers (indicated by black arrows) were included in the cell count illustrated by Figure 7.10.

A

B

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7.4. Discussion

Previous studies have proposed a role for IFNγ in mediating liver regeneration following

chronic liver injury 154,157,223, but the effects of IFNγ on the HPC response remain

undefined. It was the purpose of this study to elucidate the effects of IFNγ on the HPC

compartment in a model of chronic liver injury induced by CDE feeding. Following two

weeks of CDE feeding, mice administered 2ng IFNγ daily, had an exacerbated

inflammatory response that was accompanied by an increased HPC response and greater

degree of fibrosis compared to placebo.

IFNγ treatment also appeared to promote apoptosis of hepatocytes while leaving the HPC

population largely unchanged. This supports the previous finding by Brooling and

colleagues that IFNγ exerts a differential effect on hepatocytes and HPC 224. Findings

presented in this chapter also indicate that IFNγ may facilitate the activation of the HPC

compartment by inducing hepatocyte cell death and impairing the regenerative capacity

of the liver. Additionally, hepatocyte cell death appeared to peak at day 3 and gradually

decreased thereafter. By day 7, there was no significant difference between numbers of

TUNEL-positive hepatocytes in IFNγ-treated animals versus placebo in both CDE and

control groups, suggesting that IFNγ may only induce hepatocyte cell death transiently.

Hepatic cytokine profiles showed that IFNγ production was reduced in the IFNγ-treated

animals, thus suggesting that a negative-feedback regulatory loop exists to maintain some

degree of homeostasis. Not only were hepatic levels of IFNγ mRNA reduced by cytokine

treatment, but IL-6 and TNFα mRNA levels were also elevated in the IFNγ-treated

animals, indicative of their roles in facilitating an IFNγ-induced HPC response. However,

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the mechanism underlying the ability of this combination of cytokines to induce a HPC

response independent of liver damage caused by CDE feeding remains unknown.

Nevertheless, the observations from this study are consistent with the currently accepted

view that the extent of progenitor cell response increases with hepatocyte damage and the

liver’s inability to regenerate via hepatocyte proliferation.

IFNγ treatment in CDE fed animals exacerbated liver injury as evidenced by greater ALT

elevation, inflammation, fibrosis, morbidity and mortality. ALT exhibited a transient

elevation which returned to baseline levels by day 7. This pattern of transient response to

IFNγ treatment was also seen in the influx of inflammatory cells to the periportal regions

of the liver where HPC are thought to originate. CD45 immunohistochemical staining of

initial 14 day timepoint samples showed that IFNγ caused up to a 4-fold increase in

inflammatory cells. In addition, clusters of inflammatory cells were seen in the

control/IFNγ group at Day 3, to a lesser extent Day 5. By Day 7 this phenotype was no

longer apparent. This demonstrates that IFNγ treatment alone can cause a transient influx

of inflammatory cells, but in the background of the CDE diet IFNγ can greatly increase

inflammation which then leads to more fibrosis. It is also possible that the cytokines

released by inflammatory cells in the periportal region play pivotal roles in (i) activation

of the HPC compartment, (ii) causing hepatocyte damage, and (iii) initiating fibrogenesis.

Inflammatory cells have been shown to release IL-6, TNFα, LTβ and IFNγ – cytokines

shown to modulate the hepatic progenitor cell response. IL-6 has been shown to have a

mitogenic effect on HPC 167,168,282. Secreted IL-6 binds to its receptor’s α-chain (gp80)

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which then forms a complex with the gp130 receptor thus activating the JAK-STAT

pathway. TNFα signals through interaction between ligand and its two receptors TNFR1

and TNFR2, which are expressed on the surface of Kupffer cells. This activates NF-κB

downstream, which plays an important role in progenitor cell proliferation and

differentiation 249. While hepatocytes are usually resistant to TNFα-induced toxicity, they

may undergo TNFα-induced apoptosis in the setting of selective inhibition of NF-κB,

which is responsible for the upregulation of genes responsible for cell survival 294,295.

Often, inhibition of NF-κB is attained through the sustained activation of JNK and c-jun,

as well as increased transcriptional activity of transcription activator protein(AP-1), thus

sensitizing hepatocytes to TNFα-mediated apoptosis 296. Additionally, IFNγ has been

shown to work synergistically with TNFα to induce apoptosis 297,298.

Treatment with IL-6 has been used to successfully reverse impaired liver regeneration in

TNFR1-deficient mice, suggesting that IL-6 expression is mediated by TNFα signaling in

an autocrine or paracrine manner. TNFα appears to be equally important in the

proliferative response of progenitor cells. Studies using TNFα inhibitors and TNFR1 KO

mice have demonstrated that TNFα signaling is essential for HPC-mediated liver

regeneration 155,165. Thus the upregulation of TNFα and IL-6 expression in IFNγ treated

mice is likely to be jointly responsible for inducing proliferation of HPC. However,

elevated levels of TNFα mRNA levels in the IFNγ-treated mice are also a plausible

effector for increased hepatocyte apoptosis.

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The most unexpected outcome of this study was the increased fibrosis observed in the

IFNγ-treated animals, particularly since IFNγ has been used in experimental treatment of

liver fibrosis in both animal models and human disease 219,299. IFNγ was identified as an

inhibitor of ECM synthesis and activation of hepatic HSC by antagonizing TGFβ/Smad

signaling 300,301. Decreased mRNA levels of TGFβ showed that while IFNγ treatment was

effective in reducing TGFβ expression, this was not sufficient to reduce fibrosis. The

difference between the outcomes of this study compared to previous reports may be

attributed to the experimental feeding regime. There may exist an IFNγ-inducible

TGFβ/Smad-independent pro-fibrogenic pathway activated by CDE feeding. The CDE

diet has been reported to change cytokine production 154 hence it is also possible that

administration of IFNγ to CDE-fed mice resulted in increased fibrosis through interplay

between IFNγ and other cytokines. Additionally, we observed a marked influx of

inflammatory cells in the control/IFNγ group, suggesting that IFNγ treatment may have

primed the liver for exacerbated parenchymal damage and fibrosis mediated by TNFα.

In summary, IFNγ-treatment in the CDE model increases the magnitude of HPC

response, hepatocyte damage and liver fibrosis. Findings presented in the current chapter

suggest that IFNγ treatment increases mRNA levels of IL-6 and TNFα, contributing to

increased HPC response and hepatocyte damage in the CDE model. IFNγ production in

the liver may also be regulated by a negative feedback loop. IFNγ may prime the liver for

inflammatory/HPC response by attracting inflammatory cells to the periportal regions

while inducing fibrogenic repsonse via a TGFβ/Smad-independent pathway. Results from

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this study further support that there is a close relationship between the HPC response,

inflammation and fibrosis.

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8. General Discussion

8.1. Introduction

As the concept of HPC-mediated liver regeneration becomes more widely accepted, there

has been global interest in exploiting these otherwise dormant progenitor cells. The

precursor-product relationship between HPC and cancer has been extensively researched

in the past two decades, giving rise to an enormous number of experimental

carcinogenesis animal models focusing on HPC activation. Consequently, numerous HPC

cell lines have been developed, ranging from the heterogeneous wild-type cell lines to

those which are clonally derived.

8.2. Hepatic Progenitor Cells and Cancer

The significance of HPC in cancer research lies in the precursor-product relationship

between HPC and HCC. Chronic hepatitis C patients represent a significant proportion of

those at risk for developing HCC. While there have been studies showing that interferon

therapy reduced the risk of HCC in these patients regardless of viral clearance, there had

been little or no research performed to elucidate the mechanism of this risk reduction.

The published literature supported the genesis of the hypothesis that interferon therapy

reduced numbers of HPC, thereby reducing targets of malignant transformation and risk

of HCC development. This was confirmed by showing that interferon therapy drastically

reduced the numbers of c-kit-positive HPC in non-responding chronic hepatitis C patients

(Chapter 3). The c-kit-positive cells represent the most primitive group of HPC studied,

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suggesting that this subpopulation of HPC have greater potential for plasticity towards

differentiation compared to the more matured π-GST-positive and CK19-positive HPC.

Furthermore, IFNα reduced HPC numbers in vitro through a combination of pro-

apoptotic effects, anti-proliferative effects and stimulation of differentiation (Chapter 4).

Providing further evidence for the validity of these observations, IFNα reduced HPC

numbers in vivo in the CDE mouse model. IFNα also altered the hepatic cytokine profile,

which may have contributed to the modulation of the HPC response (Chapter 5). These

findings provide some insights into possible mechanisms by which interferon therapy

may reduce the risk of HCC development in chronic hepatitis C patients regardless of

viral clearance 82,227,302. Risk reduction of HCC is accompanied by a decrease in serum

ALT levels, which has been suggested as a prognostic indicator of risk for HCC

development in chronic viral hepatitis 79,303-305. The reduction of HPC numbers mediated

by IFNα was not accompanied by a reduction in fibrosis, although there was a reduction

in numbers of activated HSC in IFNα-treated animals. Once again, this emphasizes the

close link between HSC and HPC populations. It is likely that reduction in fibrosis would

have been observed if IFNα treatment was extended since HSC activation has been

identified as a key effector of hepatic fibrogenesis 306,307.

Chapters 3 to 5 are the first studies to show a relationship between reduction of HPC

numbers and interferon treatment in chronic hepatitis C patients. On this basis it is

proposed that the mechanism through which this occurs is via a combination of effects on

modulating cytokines involved in the HPC response, as well as cellular processes of

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differentiation, apoptosis and proliferation. Interestingly, the animal studies revealed a

concurrent reduction in HPC and HSC numbers as a result of IFNα treatment –

supporting the currently proposed theory of Clouston and colleagues that the two cell

types interact to shape the outcome of HPC-mediated liver regeneration 289. Several

research groups have noted the interplay between inflammation, fibrosis and the HPC

response resulting in recent publications on the relationship between inflammatory

cytokines, HSC and HPC 42,154,156,157,282,308. There is a close anatomic relationship

between the two cell types during HPC-mediated liver regeneration and it has been

previously suggested that this may be due to production of growth factors and expression

of receptors by these cells during the period of restitution 42. Additionally, IFNα

treatment caused significant changes in the hepatic cytokine profile. This change in

cytokine levels has been shown to modulate numbers of HPC in vitro and the magnitude

of the HPC response in vivo 154-157,168.

8.2. Interferon γ Modulates the Hepatic Progenitor Cell

Response

Previous studies have sought to determine the effects of cytokines on the HPC response.

A study by Knight and colleagues involving TNFR1 knockout mice showed that lack of

TNFα signaling greatly reduces HPC proliferation and thus HPC response during the

preneoplastic phase of hepatocarcinogenesis 155. TNFα is an upstream inducer of IL-6

expression during acute liver regeneration, thus it is not surprising that inhibition of the

two cytokines by dexamethasone also results in a diminished HPC response 167. Other

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members belonging to the TNF family including TNF-like weak inducer of apoptosis

(TWEAK) and LTβ have been implicated in HPC-mediated liver regeneration. TWEAK

appears to have a selective mitogenic effect on HPC 309, while LTβ has been shown to be

crucial for liver regeneration during chronic liver injury, and its activation is regulated by

IL-6 and IL-1β 282.

IFNγ knockout mice have an impaired HPC response with marked reduction in numbers

of MPK-positive HPC following administration of the CDE diet 157. The IFNγ gene

network has also been shown to be of particular importance in HPC-mediated liver

regeneration 223. Thus, the later portion of this thesis was dedicated to the study of IFNγ

and the HPC response. IFNγ is responsible for coordinating a wide range of cellular

programs through transcriptional regulation of immunologically relevant genes.

Experimental treatment of viral hepatitis with IFNγ has yielded promising results 219,299.

The antifibrotic effects of IFNγ have been attributed to its antagonistic effects on

TGFβ/Smad signaling pathway, and inhibition of HSC activation 301,310,311.

The in vitro studies (Chapter 6) demonstrated that IFNγ had pro-apoptotic and anti-

proliferative effects on the PIL cell lines. These results were also reproduced in primary

cell cultures. Our findings differ from those of Brooling and colleagues although it should

be noted that the LE cell lines used by their group were derived from rats whereas the

PIL cell lines used in the current work were derived from mice 224. As previously

mentioned, there are several possible explanations for these apparently contradictory

findings. The culture conditions used for the LE and PIL cell line experiments were

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different. Supplementation of culture media with fetal calf serum used for maintenance of

LE cell lines may have affected the impact of IFNγ treatment of the cells. On the other

hand, PIL cells were first serum starved and then cultured in serum-free media to ensure

that any changes in apoptotic and/or proliferative status could be reliably attributed to

IFNγ treatment. It is also important to note that pro-proliferative effects of IFNγ on LE

cells were only seen when combined with LPS or TNFα.

Chapter 7 describes attempts to confirm and extend the in vitro observations in the whole

animal model. Surprisingly, IFNγ not only increased the magnitude of the HPC response

but also increased the extent of morbidity and mortality in the CDE-fed mice. During the

early stages of carcinogenesis, small pockets of inflammatory cells were observed in the

IFNγ-treated mice regardless of CDE feeding. IFNγ is known to affect leukocyte

trafficking – a process which it regulates by altering expression of chemokines and

adhesion molecules (For review, see200). The current findings therefore suggest that IFNγ

primes the liver for an inflammatory response by recruiting inflammatory cells. This

could explain the exacerbated inflammation, parenchymal damage, fibrosis and HPC

response seen in the IFNγ/CDE animals. Therefore, results presented in Chapter 7 argue

against a possible use of IFNγ for treatment of fibrosis, suggesting that IFNγ may worsen

fibrosis and exacerbate inflammatory events, as well as increase the numbers of HPC,

thereby increasing the risk of HCC development. Previous work from our group has

shown that CDE feeding triggers a series of cytokine events 154 which may be responsible

for interacting with the IFNγ treatment and giving rise to the resulting fibrotic and

inflammatory changes that precede and precipitate the HPC response. An in-depth study

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into the interaction between IFNγ and other cytokines induced by CDE feeding and their

combined effect on the HPC response might clarify which combination of these factors

override the anti-fibrotic effects of IFNγ resulting in greater devastation.

8.3. Concluding Remarks

The findings documented in this thesis raise several interesting questions. Numbers of

HPC could be controlled with IFNα treatment, thereby suggesting that chronic hepatitis C

patients who fail to respond virologically (by clearing the virus) or biochemically

(through decreased serum ALT levels) during IFNα therapy may still stand to gain from

treatment. With an estimated 250,000 Australians currently infected with hepatitis C

virus, and an additional 10, 000 new infections per year and the majority of these

individuals at risk of chronic infection and HCC, IFNα therapy may have great potential

to impact on future strategies to control the development of HCC.

Significantly, IFNγ could, under certain conditions, exacerbate inflammation, fibrosis and

the HPC response. These observations highlight the need for caution when considering

IFNγ as a possible treatment of fibrosis as suggested by some studies. While the potential

therapeutic effects of IFNγ cannot be totally excluded, it is important to further

investigate the interaction between IFNγ and other inflammatory cytokines.

Another emerging issue pursued with growing interest is the concept of cancer stem cells.

Chiba and colleagues recently reported cancer stem cells expressing AFP and CK19 in

half of the human cancer cell lines studied, and proposed that these cells exist within the

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upstream hierarchy of HCC and retain the latent ability to differentiate 312. The presence

of cancer stem cells in the liver raises the possibility of differentiation therapy for

treatment of HCC, or at least for the control of HPC in patients at risk of developing

HCC. Findings presented in this thesis suggest that IFNα is a likely candidate, and its

effect on the molecular events pivotal to HPC differentiation warrant further

investigation, particularly since this cytokine has been used for adjuvant therapy of

melanoma in the past, owing to its ability to induce differentiation 313,314.

A recent paper published in Oncogene outlined new therapies for HCC and suggested

that in addition to differentiation therapy and kinase inhibitors, gene and cell therapy

could also be used for treatment of HCC 315. Whilst gene therapy is currently in its

infancy, it could likely be delivered through the transduction of viral vectors bearing a

transgene which facilitates the production of therapeutic substances inside the tumor

mass while keeping systemic levels low. Already fibroblasts transduced with

retroviruses-encoding IFNα have been used for experimental treatment of ovarian

carcinoma 316. At this juncture, the first generation viral vectors do not reach clinical

efficacy. However, lessons learnt from pioneer trials, use of Positron Emission

Tomography (PET) for monitoring gene expression and further development of viral

vectors may well lead to gene and cell therapy becoming the new faces of cancer

treatment.

In conclusion, we live in an exciting time for cancer research. As our paradigm shifts

towards more specific and personalized cancer treatment, and we increase our

Chapter 8

154

understanding of cancer stem cells at the cellular and molecular level, we take one step

closer to realizing the dream shared by all – of making cancer a disease of the past.

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155

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