TARGETED DELETIO OF FIBRI OGE LIKE PROTEI 2 (FGL2) E HA CES IMMU ITY I A MURI E MODEL OF ACUTE VIRAL HEPATITIS CAUSED BY LYMPHOCYTIC CHORIOME I GITIS VIRUS (LCMV)
By
Ramzi Khattar
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Immunology
University of Toronto
© Copyright by Ramzi Khattar 2011
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TARGETED DELETIO OF FIBRI OGE LIKE PROTEI 2 (FGL2) E HA CES IMMU ITY I A MURI E MODEL OF ACUTE VIRAL HEPATITIS CAUSED BY LYMPHOCYTIC CHORIOME I GITIS VIRUS (LCMV)
Master of Science, 2011 Ramzi Khattar Department of Immunology University of Toronto
ABSTRACT
Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the
FGL2 FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.
ii ACK OWLEDGEME TS
I would like to thank my supervisor Dr. Gary Levy and members of my supervisory committee, Dr. Michael Ratcliffe, Dr. Pamela Ohashi and Dr. Reginald
Gorczynski for their continued guidance and support.
I acknowledge the contributions of Dr. Lang and the laboratory of Dr. Pamela
Ohashi for their expertise and consultation in the LCMV model; Dr. Phillips and Dr
Adeyi for their assessment of liver pathology; Dr. Shalev and Dr. Selzner for their expertise in liver disease. I would also like to acknowledge Natalie Yavorska and Darrin
Gao for providing their assistance with many of the experiments.
My time in the laboratory of Dr. Gary Levy has been especially enriched by the current and past members of the IBDL and research laboratory, especially Cheryl Bodnar,
Justin Manuel, Jianhua Zhang, Agata Bartczak, Peter Urbanellis, Wendy Shyu, Olga Luft,
Charmaine Beal, Anna Cocco, David Smookler and Ashley Lau.
I would especially like to thank my family (Emile Khattar, Leila Khattar, Hani
Khattar, Natasha Khattar, Randa Manoukian and Andre Manoukian) friends (Tim Tsui,
Henry Biggs, Gregory Rassias, Jonathan Dickens, Chia Barsen, Terry Tai, Alice Ng, and
Rashedul Amin) and colleagues from the department of immunology for their support and encouragement. I would also like to thank the employees at Starbucks café (York
Mills and Leslie) for their help and support over the 11 years I have been studying there.
iii TABLE OF CO TE TS
Abstract ii Acknowledgements iii Table of Contents iv vii List of Tables vi List of Figures vi vii List of Abbreviations vii x Contributions to Science xi
Chapter I I TRODUCTIO 1 27 I.1 Viral Hepatitis 1 4 I.1.1 Hepatitis C Viral Infection 3 I.1.2 Current Treatments for HCV infection 4
I.2 The Immune Response 4 14 I.2.1 Innate Immunity 6 I.2.2 Innate Immune Recognition 7 I.2.3 Adaptive Immunity 9 I.2.4 Cellular Immunity 9 I.2.5 Humoral Immunity 11 I.2.6 Immune Homeostasis 13
I.3 Mechanisms of Evading Immune Recognition and Silencing the Immune Response in HCV Infection 14 16 I.3.1 Treg and HCV Infection 15
I.4 Fibrinogen like protein 2 (FGL2) 17 21 I.4.1 Membrane bound FGL2 18 I.4.2 Secreted FGL2 20
I.5 Lymphocytic Choriomeningitis Virus (LCMV) 22 25 I.5.1 LCMV WE 23
I. 6 Hypothesis 25 27
Chapter II MATERIALS A D METHODS 28 36 II.1 Mice 28 II.2 Virus 28 II.3 Cell Culture Media 29 II.4 Synthetic Peptides 29 II.5 Blood collection 29 II.6 Measurement of Alanine Aminotransferase (ALT) 29
iv II.7 Measurement of Plasma FGL2 29 II.8 Isolation of Lymphocytes from Spleens and Lymph Nodes 31 II.9 Isolation of Intrahepatic Mononuclear Cells 31 II.10 Histology and Immunohistochemistry 32 II.11 Morphometric Analysis 32 II.12 Chromozym Th assay 32 II.13 In Vitro Infection of Fibroblasts with LCMV in the Presence of FGL2 33 II.14 Flow Cytometry 34 II.15 Assessing LCMV Specific Humoral Responses 34 II.16 Measurment of Neutralizing Antibody 35 II.17 Intracellular Cytokine Analysis 36 II.18 Statistical analysis 36
Chapter III RESULTS 37 56
III.1 The Prothrombinase Activity of FGL2 Prevents Early Viral Dissemination in Wild Type Mice 37
III.2 Secreted FGL2 is Induced Following LCMV Infection of Wild Type Mice: 40
III.3 Targeted Deletion of fgl2 Results in Enhanced Intrahepatic Antiviral Activity Following LCMV Infection: 41
III.4 Dendritic Cells Isolated From fgl2 / Mice Have Increased Activity Following Infection with LCMV WE: 45
III.5 Targeted Deletion of fgl2 Enhances the Frequency of Virus Specific T Cells and Increases Responsiveness to Ex Vivo Peptide Restimulation Following LCMV WE: 46
III.6 Targeted Deletion of FGL2 Enhances Humoral Responses Towards LCMV: 51
III.7 Targeted Deletion of fgl2 Enhances Immunity to LCMV Upon in Secondary Reinfection: 53
III.8 Conclusions: 55
Chapter IV DISCUSSIO 57 70
IV.1 Future Directions 69
v
Chapter V REFERE CES 71 91
LIST OF TABLES
Introduction
Table I 1 The immune response is composed of both innate and adaptive components 6
Table I 2 Route of inoculation and strain of LCMV affect disease manifestation and clearance kinetics. 23
Table I 3 Comparing the clinical outcomes of infection with different strains of LCMV with chronic HCV infection 24
LIST OF FIGURES
Introduction
Figure I 1 Mechanisms for clearance of HCV: 12
Figure I 2 Treg inhibit the adaptive immune response to HCV: 16
Figure I 3 The contribution of FGL2 to innate and adaptive immunity 18
Figure I 4 Proposed model for how FGL2 affects the early phase of viral infection 26
Figure I 5 Proposed model for how FGL2 affects the adaptive immune response to viral pathogens 27 Results
Figure III 1.1 FGL2 prothrombinase activity is induced in macrophages upon LCMV infection 38
Figure III 1.2 Fgl2 / mice have enhanced viral replication prior to the induction of adaptive immunity 38
Figure III 1.3 FGL2 does not mediate its protective effects by globally inhibiting viral entry or production 39
Figure III 2.1 FGL2 is induced in vivo upon LCMV WE infection and
vi remains elevated long after viral clearance from the liver 41
Figure III 3.1 Targeted deletion of fgl2 results in enhanced inflammatory responses within the liver towards LCMV infection 43
Figure III 3.2 Fgl2 / mice and wild type mice have similar viral clearance kinetics, despite enhanced early viral replication in fgl2 / mice 44
Figure III 4.1 Fgl2 / mice have enhanced DC activation at day 1 following infection with LCMV WE 45
Figure III 5.1 Fgl2 / mice have increased frequencies of intrahepatic virus specific CD8 + T cells following infection with LCMV WE 46
Figure III 5.2 Fgl2 / mice have increased frequencies of CD8 + T cells responding to peptide restimulation following LCMV infection 49
Figure III 5.3 Fgl2 / mice have increased frequencies of CD4 + T cells responding to peptide restimulation following LCMV infection 50
Figure III 6.1 Fgl2 / mice have increased humoral responses towards LCMV 52
Figure III 7.1 Fgl2 / mice are completely protected in secondary reinfection with LCMV WE 54
Figure III 7.2 Fgl2 / mice have increased frequencies of CD8 + T cells responding to peptide restimulation following LCMV reinfection 55
Discussion
Figure IV 1 Histopathological and biochemical features of LCMV Cl13 infection in wild type mice 64
Figure IV 2 Viral titres in wild type mice following LCMV Cl13 infection 66
Figure IV 3 FGL2 is induced in vivo upon LCMV Cl13 infection and remains persistently elevated 67
Figure IV 4 Mean plasma levels of FGL2 in patients with chronic HCV infection 68
vii LIST OF ABBREVIATIO S
2’5’ OAS 2’5’ Oligoadenylate Synthetase α DG α Dystroglycan Ab Antibody AICD Activation Induced Cell Death ALT Alanine Transaminase APC Antigen Presenting Cell APC Allophycocyanin ARM Armstrong BCR B cell Receptor BSA Bovine Serum Albumin CD Cluster of Differentiation CHO Chinese Hamster Ovary CINC Cytokine Induced Neutrophil Chemoattractant Protein Cl 13 Clone 13 CTL Cytotoxic T Lymphocyte CTLA4 Cytotoxic T lymphocyte Antigen 4 DAB 3,3’ Diaminobenzidine DAMP Danger Associated Molecular Patterns DC Dendritic Cells DMEM Dulbecco’s Modified Eagle Medium DN T cells Double Negative T cells dsRNA Double Stranded Ribonucleic Acid ECM Extracellular Matrix EDTA Ethylenediaminetetraacetic acid ELISA Enzyme Linked Immunosorbant Assay FCγRIIB Fragment of Crystallization γ Receptor IIB FCS Fetal Calf Serum FGL2 Fibrinogen Like Protein 2 fgl2 / Fibrinogen Like Protein 2 Knockout FITC Fluorescein isothiocyanate FRED Fibrinogen Related Domain FVH Fulmanent Viral Hepatitis b GP 33 41 H2 D (Peptide) KAVYNFATC b GP 61 80 I A (Peptide) GLNGPDIYKGVYQFKSVEFD H&E Hematoxylin and Eosin HAV Hepatitis A Virus HBV Hepatitis B Virus HBSS Hanks Balanced Salt Solution HCC Hepatocellular Carcinoma HCV Hepatitis C Virus HDV Hepatitis D Virus HEV Hepatitis E Virus HIV Human Immunodeficiency Virus
viii HRP Horseraddish Peroxydase IC Intracranially IFN γ Interferon γ Ig Immunoglobulin IL2 Interleukin 2 IL4 Interleukin 4 IL5 Interleukin 5 IL6 Interleukin 6 IL7 Interleukin 7 IL7R Interleukin 7 Receptor IL10 Interleukin 10 IL13 Interleukin 13 IP Intraperitoneally IV Intravenously LCMV Lymphocytic Choriomeningitis Virus L Glut L Glutamine LPS Lipopolysaccharide L Segment Large Segment MCP 1 Monocyte Chemoattractant Protein 1 MFI Median Fluorescence Intensity MHC Major Histocompatibility Complex MHV3 Mouse Hepatitis Virus 3 MOI Multiplicity of Infection nAb Neutralizing Antibody NFκB Nuclear Factor Kappa Light Chain Enhancer of Activated B cells NK Natural Killer NP Nucleocapsid b P 396 404 H2 D Peptide (FQPQNGQFI) OCI Ontario Cancer Institute PAMP Pathogen Associated Molecular Pattern PBS Phosphate Buffered Saline PD1 Programmed Death 1 PEG IFNα Pegylated Interferon α PE Phycoerythrin PE CY5.5 Phycoerythrin Cyanine 5.5 PEM Peritoneal Exudate Macrophages PI Post Infection PKR Protein Kinase R PRR Pattern Recognition Receptor RBC Red Blood Cell RIG I Retinoic Acid Inducible Gene I RPMI 1640 Roswell Park Memorial Institute 1640 medium SARS Severe Acute Respiratory Syndrome SC Subcutaneous SEM Standard Error Mean SI RNA Small Interfering RNA
ix SPF Specific Pathogen Free S Segment Small Segment SVR Sustained Virologic Response TCR T cell Receptor TH1 T helper cells 1 TH2 T helper cells 2 TLR Toll like Receptor TMB Tetramethyl Benzidine TNF α Tumor Necrosis Factor α Treg Regulatory T cells TTBS Tween Tris Buffered Saline
x CO TRIBUTIO S TO SCIE CE
PUBLICATIO S:
Ma, X.Z., Zhang, J., Bartczak, A., Khattar, R. , Chen, L., Liu, M.F., Edwards, A., Levy, G.A., McGilvray, I.D. Proteasome inhibition in vivo promotes survival in a lethal murine model of SARS. J. Virol. 2010 84(23), 12419 12428.
Foerester, K., Zhu, Y. Helmy, A., Khattar, R. , Adeyi, O.A, Wong, K.M., Shalev, I., Clark, D.A., Wong, P., Heathcote, E.J., Phillips, M.J., Grant, D.R., Renner, E.L., Levy, G.A., Selzner N. The Novel Immunoregulatory Molecule FGL2: A Potential Biomarker for Severity of Chronic Hepatitis C Infection. J Hepatology. 2010. 53 (4), 608 615.
CO FERE CES:
Khattar, R. , Yavorska, N., Selzner, N., Shalev, I., Phillips, J., Adeyi, O., Bartczak, A., Urbanellis, P., Zhang, J., Manuel, J., Levy G.A. FGL2 inhibits the adaptive immune response in a model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus. American Association for the Study of Liver Diseases . San Francisco, California. (Oral – November 4 8 2011).
Khattar, R. , Selzner, N., Shalev, I., Phillips, J., Adeyi, O., Bartczak, A., Urbanellis, P., Zhang, J., Manuel, J., Levy G.A. FGL2 a Novel Treg Effector Molecule Plays a Critical Role in Regulating Immune Responses to Lymphocytic Choriomeningitis Virus Infection. International Liver Transplantation Society. Valencia, Spain. (Poster – June 24, 2011).
Khattar, R. , Yavorska, N., Selzner, N., Shalev, I., Phillips, J., Adeyi, O., Bartczak, A., Urbanellis, P., Zhang, J., Manuel, J., Levy G.A. Fibrinogen like Protein 2 (FGL2) is a Negative Regulator of Inflammatory Responses Within the Liver Toward Lymphocytic Choriomeningitis Virus (LCMV) Infection. The Program in Regenerative Medicine: Annual Regenerative Medicine Symposium. Toronto, Ontario (Oral – April 6, 2011).
Khattar, R. , Selzner, N., Shalev, I., Phillips, M.J., Adeyi, O., Bartczak, A., Urbanellis, P., Zhang, J., Manuel, J., Levy G.A. Fibrinogen like Protein 2 (FGL2) is a Critical Mediator in Regulating Inflammatory Responses Within the Liver Toward Lymphocytic Choriomenigitis Virus (LCMV) Infection. American Association for the Study of Liver Diseases . Boston, Massachusetts. (Poster October 30 – November 3 2010). President’s Choice Poster Award.
Khattar R., Selzner, N., Zhu, Y., Shalev, I. , Bartczak, A., Urbanellis, P., Levy G.A. Fibrinogen Like Protein 2 is a Predictive Indicator of Liver Pathology and Disease Progression in Patients with Chronic Hepatitis C Infections. Ontario Quebec Undergraduate Immunology Conference. Toronto, Ontario. (Oral May 6 May 7 2008).
xi CHAPTER I
I TRODUCTIO
I.1 Viral Hepatitis:
Viral hepatitis is a serious health problem affecting over 500 million individuals world wide. The etiological agents of viral hepatitis consist of 5 genetically unrelated viruses that infect and replicate within the hepatocyte, causing an immune mediated inflammation of the liver 1 5. The interactions that occur between the host’s immune response towards the virus and the mechanisms employed by the virus to evade immune recognition often dictate the consequences of infection. Therefore, infection by hepatotropic viruses can result in asymptomatic infection, severe acute infection that can result in death, persistent life long infection or clearance with long term immunity.
In adult immunocompetent individuals, infections with hepatitis A virus (HAV), hepatitis B virus (HBV) or hepatitis E virus (HEV) often results in viral clearance with mild symptoms of jaundice, hepatomegaly and abdominal discomfort 2 5. For such patients, the immune system recognizes the virus and mounts an appropriate immune response resulting in viral clearance. A small proportion of patients infected with HAV or
HBV do not control viral replication early in infection and develop a life threatening condition called fulminant viral hepatitis (FVH) 2 3. Highlighting the importance of host virus interactions, patients with FVH generate an inappropriately strong immune response to the virus that results in organ failure and ultimately death. Fortunately, vaccination strategies have proven effective in preventing the transmission of HAV and HBV, which has consequently reduced the incidence of FVH.
1 The most challenging problems that arise in the medical management of hepatotropic viral infections occur in persistent infection. Due to a heightened propensity towards immune tolerance in neonatal life, 90 % of HBV neonatal infections that occur through vertical transmission result in persistent life long infection 2. For this reason, 350 million individuals are persistently infected with HBV. Hepatitis D viral (HDV) has evolved to utilize the replication machinery of HBV and exclusively occurs in the context of persistent HBV infection. Consequently, HBV/HDV co infection results in a 5 20 % increased risk of liver failure 4. The long term sequelae of chronic HBV infection or
HBV/HDV co infection are cirrhosis and hepatocellular carcinoma (HCC). Through the use of perinatal passive immunization however, the incidence of vertical transmission to a neonate is reduced significantly 6.
Although such strategies have been critical in preventing the transmission of
HBV, vaccination does not benefit previously infected individuals. Moreover, there are currently no effective treatments to prevent the transmission of hepatitis C virus (HCV), which result in persistent infection in 85 % of individuals 7. Due to the high prevalence of persistent infection, many researchers study HCV to identify the factors that subdue the immune response and consequently establish chronicity. However, the lack of an appropriate small animal system and cell culture model susceptible to infection has hindered the characterization of such immunosuppressive factors 8. Understanding the mechanisms that hepatotropic viruses employ to attenuate the immune response will be critical in the development of novel therapeutics to treat patients with chronic hepatitis virus infection.
2 I.1.1 Hepatitis C Viral Infection:
With an estimated 170 million individuals infected chronically world wide, HCV infections represent a significant epidemiological and economic burden on society 1. Upon exposure to infected blood, some individuals mount a robust innate and adaptive immune response resulting in spontaneous clearance, with little complications of infection 7.
However, 85 % of individuals exposed to HCV develop a life long viremia with chronic infection and will require a liver transplant. In fact, due to the prevalence of chronic HCV infections in the United States, HCV was found to be the cause of 40 % of all chronic liver diseases and a leading indication for liver transplants 1. Often accompanying the chronic liver infection are profound changes in liver architecture, due to a continual low grade activation of the immune response. The secretion of a number of inflammatory mediators and growth factors both by parenchymal and nonparenchymal liver cells in response to HCV antigen can activate stellate cells, myofibroblasts and fibroblasts to initiate the deposition of extracellular matrix (ECM) proteins 9. The progressive fibrotic remodeling of the liver architecture replaces functional liver tissue with scar tissue and disrupts the exchange of nutrients and waste products with the blood supplying the liver.
10 15 % of chronically infected patients develop decompensated cirrhosis of the liver, with the development of ascites, the emergence of esophageal varices, portal hypertension and encephalopathy 7. Following the diagnosis of decompensated cirrhosis, a patient’s 5 year survival rate drops to 50 % 10 . Another frequent consequence of persistent
HCV infection is the development of HCC, due to a combination of chronic injury and mutagenic inflammation to drive transformation.
3 I.1.2 Current Treatments for HCV infection:
Due to the regenerative capacity of the liver, antiviral treatments that control HCV replication have the capacity to restore liver functionality and limit the progression to cirrhosis and HCC. The current treatment for HCV consists of an antiviral drug regime of pegylated interferon alpha (PEG IFNα) and Ribavirin. PEG IFNα is a polyethylene glycol derivative of natural type I IFN with improved efficacy as an antiviral agent. Type
I IFN inhibits viral replication by initiating the transcription of many antiviral genes including, 2’5’ oligoadenylate synthetase (2’5’ OAS) and protein kinase R (PKR), which respectively work to disrupt global transcription and translation within the virally infected cell 11 . Ribavirin is a nucleoside analogue that can inhibit the replication of HCV at multiple levels. Ribavirin can bias naive CD4 + T helper cell lineage commitment towards a TH1 lineage, with enhanced expression of IFN γ and greater production of TH1 IgG2a antibody by plasma cells. The enhanced cellular immune response mediates the protective antiviral activity of ribavirin 11 12 . Ribavirin can also enter cells to inhibit the production of guanosine phosphates and introduce mutations within the viral genome 13 .
These antiviral medications are effective at reducing viral replication, but only 50% of patients attain a sustained virologic response (SVR). New agents including the protease inhibitors telaprevir and boceprevir 14 15 are being studied and results to date suggest that in combination with current therapy, response rates are enhanced. Despite the improvements in current treatment options, a large proportion of patients remain unresponsive to therapy. There is a pressing need to understand the factors that prompt responsiveness to antiviral therapy and progression to chronic infection.
4 I.2 The Immune Response:
The integration of both innate and adaptive arms of the immune response is required for clearance of HCV infection. The immune system is comprised of cells and effector molecules that protect the body from infection with pathogens. To protect its host from pathogens, the immune response has evolved to recognize a universal repertoire that is, in a non pathological state, capable of discriminating self from non self.
Such diversity is maintained somatically after embryogenesis, as cells mature to become effector cells. Thus, identical twins will not have identical immune repertoires.
Importantly, the cells that exit to the periphery, following maturation, must be self tolerant to prevent autoimmunity. To achieve tolerance to self, auto reactive cells die during development through a process called central tolerance. Auto reactive lymphocytes can also be tolerized in the periphery, if such central tolerizing mechanisms fail to prevent the expansion of auto reactive lymphocytes. Consequently, the immune response has evolved to detect foreignness and to produce a protective response against that foreignness. The immune response possesses both innate germ line encoded components and adaptive components that serve to protect the host organism from invasive pathogens ( Table 1 1).
5
Table I 1. The immune response is composed of both innate and adaptive components: Innate immunity limits the entry or early replication of invading pathogens by providing a physical barrier to entry or by recognizing pathogens in the context of broadly specific germline encoded receptors. Adaptive immunity serves to control the replication of an invading pathogen if innate immunity fails by recognizing pathogens in the context of specific receptors.
I.2.1 Innate Immunity:
In viral infection, the innate immune response represents the first barrier to infection. Innate components of the immune system are those that require no prior sensitization or clonal expansion to exert their protective effects. For example, the epithelium not only provides a mechanical barrier that prevents the entry of the virus into the body, but is also lined with mucosal secretions containing anti microbial peptides 16, nucleases 17 and mucosal IgA 18 that interfere with infection. If these mechanical barriers fail to prevent infection, innate systems such as, complement 19 , NK cell mediated lysis 20 and innate antibody production by B 1 cells 21 can work to suppress overwhelming viral
6 replication. In HCV infection, innate antiviral responses do not significantly contribute to early viral control, but remain critical in the initiation of the inflammatory response and the recruitment of leukocytes to the liver 22 24 . Phagocytosis by neutrophils, macrophages and dendritic cells (DC) represents a critical step in sensing HCV infection to initiate a specific adaptive immune response.
I.2.2 Innate Immune Recognition:
Both phagocytes and infected hapatocytes are involved in innate immune recognition of HCV. The stimuli that initiate an immune response towards viral pathogens may occur in the recognition of pathogen associated molecular patterns
(PAMP) expressed by the virus, as proposed by Janeway’s “stranger model” of immune recognition 25 . This model suggests that immune responses are elicited towards pathogens through the recognition by pattern recognition receptors (PRR) of well conserved PAMP expressed exclusively in the microbial world. Cytoplasmic PRR such as, RIG I26 and endosomal PRR such as, TLR3 27 are critical in the innate immune recognition of HCV double stranded ribonucleic acid (dsRNA) replicative intermediates. The stimuli that are recognized by the immune system to drive immunity towards a pathogen may also be endogenous. These are derived from endogenous proteins that are normally not exposed in the absence of tissue damage as suggested by Matzinger’s “danger model” of immune recognition 28 . The products of stressed or injured cells that are recognized by the innate immune system are termed, danger associated molecular patterns (DAMP). Consistent with this hypothesis, allografts following transplantation that are devoid of any known microbial pathogens elicit very strong immune responses against the MHC disparate donors 10 .
7 Following activation by an infectious agent or cellular necrosis, PRR send signals to initiate the transcription of innate molecules that promote inflammation 29 . The transcription of many proinflammatory cytokines is regulated by the transcription factor,
NFκB, which is normally sequestered in the cytoplasm by IκB. The signal cascades that are initiated as a result of PRR engagement promote the degradation of IκB, which unmasks a nuclear localization signal in NFκB 30 . Consequently, NFκB translocates to the nucleus and initiates the transcription of a number of molecules, such as the IFNα/β 32 , proinflammatory cytokines 33 and chemokines 34 that serve to limit the infection, activate immune cells, and attract leukocytes to the site of infection. In HCV infection, IFNα/β is induced during the acute phase of infection 35 37 .However, HCV structural and nonstructural proteins, such as E2, NS3 and NS5A directly inhibit many of the antiviral gene products, reducing their efficiency in the early antiviral response 38 41 . Inflammatory cytokines including Tumor Necrosis Factor α (TNF α) and Interferon γ (IFN γ), also increase vascular epithelial permeability to promote the exchange of proteins, cells and antigens from the site of inflammation with the draining lymphatics 42 . The inflammatory response also enhances the activity of phagocytes to further promote the phagocytosis of virus at that site of inflammation 43 44 .
Another important consequence of PRR engagement is to promote the maturation of DC, characterized by the enhanced expression of MHCII and important costimulatory molecules including, CD80 and CD86 45 . Following activation, DC will also express chemokine receptors that attract them to the lymphatics, which subsequently drain into secondary lymphoid organs, such as the spleen or lymph nodes.
8 I.2.3 Adaptive Immunity:
Although the majority of infections are prevented through innate immune mechanisms, HCV requires the induction of an adaptive immune response to achieve control. Adaptive immune responses occur through the clonal expansion of rare populations of antigen specific T or B cells.
Mature DC that have captured virus from sites of infection will digest the captured antigen into peptides and display such peptides in the context of the major histocompatibility complex (MHC). In order to initiate a robust T cell response, a T cell must recognize the peptide presented in the context of MHC. Recognition through the T cell receptor (TCR) alone however, is insufficient to fully initiate a proliferative response in antigen specific T cells. A T cell must also receive appropriate costimulation through the interaction of CD80 and CD86 expressed on mature DC with CD28 expressed on the
T cell 45 . T cells recognizing antigen on immature DC or on hepatocytes that fail to express high levels of CD80 and CD86 will become anergized 46 . Anergic T cells have limited proliferative capacity and fail to express important T cell effector molecules, such as, IFN γ and TNF α47 . Given appropriate cytokine stimulation, a T cell recognizing antigen on a mature DC will become activated and proliferate.
I.2.4 Cellular Immunity:
Following the expansion of antigen specific T cell populations, T cells exit secondary lymphoid organs and migrate along a chemokine gradient to the liver, where they can exert their effector functions in promoting immunity against HCV.
Based on their recognition of MHC, T cells are divided into two major subsets,
CD8 + T cells and CD4 + T cells. Cytotoxic CD8 + T lymphocytes (CTL) recognize
9 intracellular pathogens processed in the context of MHCI and kill target cells by secreting a number of effector molecules such as, perforin 48 and granzymes 49 . Because HCV is an obligate intracellular pathogen, CTL play a critical role both in controlling viral infection and contributing to the severity of hepatitis. For this reason, the magnitude of the hepatic
CTL response correlates with disease severity. Patients who clear the virus spontaneously or respond to interferon based therapy mount a vigorous CTL response, characterized by enhanced liver necrosis and inflammation, while patients that remain chronically infected generate a weak CTL response with limited hepatic inflammation 50 . It is the persistence of this weak inflammatory response for long periods of time that drives the progressive liver disease observed in many chronically infected HCV patients. In chimpanzee models of HCV and HBV, the histological and biochemical evidence of liver injury coincides temporally with the influx of CTL into the liver, while experimental depletion of CTL delays the onset of hepatitis, but promotes viral persistence 51 52 . Therefore, strategies to promote virus specific CTL responses can enhance viral clearance in experimental and human studies and represent a promising approach to improve the medical management of patients with viral hepatitis. CD4 + T cells, on the other hand, recognize antigen in the context of MHCII and influence the immune response through the secretion of cytokines 53. CD4 + T helper cells 1 (TH1) aide in the proliferation of CTL and secrete TH1 cytokines, such as interleukin 2 (IL 2) and IFN γ. CD4 + T helper cells 2
(TH2) aide in the maturation of B cells to promote humoral immunity and release TH2 cytokines, such as interleukin 4 (IL 4), interleukin 5 (IL 5), interleukin 6 (IL 6), interleukin 10 (IL 10) and interleukin 13 (IL 13) (Figure 1 1). Helper T cell activity is
10 lost in many chronic infections, such as HCV and HBV which often results in poor
CTL 54 57 and humoral responses 58 59 , due to a lack of appropriate cytokine stimulation.
I.2.5 Humoral Immunity:
B cells recognize specific epitopes found on whole antigens brought into secondary lymphoid organs through the lymphatics. When a B cell engages its antigen through the B cell receptor (BCR), the antigen BCR complex enters the cell through receptor mediated endocytosis. B cells will process the antigen, expressing it as peptides on their cell surface in the context of MHCII. Like CTL, B cells also require costimulation, but by CD4+ TH2 cells recognizing antigen expressed on the surface of the B cell in the context of MHCII 60 . Following activation, B cells will proliferate and differentiate into effector plasma cells and secrete antibody towards the recognized antigen.
Antibody produced by plasma cells protects against extracellular pathogens, such as bacteria and extracellular virus. Protection by antibody is mediated through a number of mechanisms 61 62 . Antibody binding to its antigen promotes phagocytosis by macrophages and DC through opsonization. Antibodies can also activate complement and enhance NK cell activity to promote lysis of the extracellular pathogens. Neutralizing antibodies directed towards the receptor binding domains of a viral pathogen can sterically hinder the interaction of the virus with its target cellular receptor 63 .
Accordingly, vaccination exposes the host to a weakened or killed variant of a specific pathogen to promote the formation of neutralizing antibodies and prevent infection with the same pathogen.
11 Characteristic of non cytopathic viral infections, the antibody response in HCV is blunted and occurs late in infection. Because of the temporal delay in the induction of humoral immunity, antibody responses do not play a critical role in controlling the replication of non cytopathic viruses 51 . Antibody depletion in experimental models of
LCMV and HBV however, causes the reemergence of virus following resolution. The reemergence of virus suggests that non cytopathic viruses are never actually cleared and a sustained neutralizing antibody response may be required for long term control 64 65 .
Figure 1 1. Mechanisms for clearance of HCV: Clearance of HCV requires the induction of a robust CTL response. CTL aid in the recognition and lysis of virally infected hepatocytes by the secretion of perforin, granzymes and antiviral IFN γ and TNF α. In order to mount a robust CTL response, a virus specific CTL must receive 2 signals. The TCR on the CTL must recognize HCV antigen presented by a DC in the context of HLAI and must receive appropriate cytokine stimulation from virus specific TH1 CD4 + T helper cells. Neutralizing antibodies induced after clearance aid in controlling replication to prevent reemergence of virus. (Figure adapted from Rosen et. al. 2008. Transplantation immunology: what the clinician needs to know for immunotherapy . Gastroenterology;134(6):1789 801 .)
12 I.2.6 Immune Homeostasis:
Following the resolution of infection, immune homeostatic mechanisms ensure that the expanded antigen specific T and plasma cell populations contract to prevent inappropriate inflammatory responses in the absence of the pathogen. One mechanism that is utilized by the immune system to limit the activity of T lymphocytes is activation induced cell death (AICD). Activation through the TCR results in the induction of apoptosis through the up regulation of death ligands such as PD 166 and FAS L67.
Activation down regulates the expression of receptors that are required to promote the maintenance of such populations, such as IL 7R 68 69 . Taken together, antigen specific lymphocytes progressively lose their effector function and die in the absence of antigenic stimulation to promote their continual maintenance and proliferation. The same processes that mediates loss of function and death of antigen specific cells in the absence of antigen also occur in T lymphocytes that have been over stimulated by a high antigenic load. In a process termed T cell exhaustion, T cells that have been over stimulated have limited proliferative capacity and are eventually deleted from the immune repertoire. T cell exhaustion is utilized by the immune system to prevent an overwhelming immunopathological response, particularly in the context of chronic infection, where antigen persistence is favored 70 72 .
Following clearance of the pathogen, a number of homeostatic mechanisms also regulate humoral immunity to prevent the pathological accumulation of antibody.
Antibody promotes the apoptosis of plasma cells by binding to the inhibitory FcγRIIB receptor expressed on mature plasma cells 73. In this negative feedback mechanism, the
13 production of antibody by plasma cells promotes the apoptosis of the plasma cells to limit the further accumulation of antibody.
Following the contraction of antigen specific lymphocytes, a small proportion of effector cells differentiate into a quiescent memory cell population. These cells survey the periphery to prevent secondary reinfection with a similar pathogen. Because of these memory cells, the secondary immune response to a pathogen occurs earlier and is greater in magnitude 74 .
I.3 Mechanisms of Evading Immune Recognition and Silencing the Immune
Response in HCV Infection:
HCV has evolved mechanisms to evade immune detection by mutational escape.
The error prone RNA dependent RNA polymerase encoded by the NS5b gene can replicate the viral genome, incorporating a large number of mutations that abrogates immune detection by mutating critical epitopes recognized by the immune response 75 .
This mechanism of viral escape makes the design of a specific vaccine to HCV difficult.
Due to the large number of mutations that occur in course of infection, HCV exists as several quasi species, rather than a single distinct species that can be targeted by a single vaccine 76 . Additionally, the high rate of mutation makes the virus extremely adaptive to the strong selective pressure exerted by the immune response to HCV 77 78 .
In addition to mutational escape, HCV silences the immune response through a number of immunomodulatory mechanisms to establish its chronicity. Patients that develop a chronic infection mount a delayed and short lived T cell response against a variety of HCV epitopes, peaking late at week 8 – 12 post infection (PI) 79 82 . In addition to the deficiencies in T cell activity, these patients also fail to produce neutralizing
14 antibodies early enough to prevent mutational escape 83 84 . Several lines of evidence have implicated regulatory T cells (Treg) in impairing both cellular and humoral immune responses to HCV 85 88 .
I.3.1 Treg and HCV Infection:
Treg are a class of immunosuppressive cells that exert regulatory function on the immune system. The immune system employs Treg to maintain self tolerance by suppressing the activation and expansion of self reactive lymphocytes that might otherwise cause autoimmune disease. Treg are also utilized to limit the magnitude of an immune response to prevent immunopathological injury that may occur in the context of infection 89 100 . Under physiological circumstances, these cells function to protect the body from the injurious actions of an overactive immune response. However, oversuppression of the immune response can occur in certain chronic infections that promote the expansion of Treg. Increased numbers of Treg are observed in patients with chronic HBV and HCV infection when compared to successfully treated patients or healthy controls 86
88 . Experimental antibody depletion of Treg enhances the immune response towards HBV and HCV 86 88, 101 105 . In vitro depletion of these cells increases virus specific T cell responsiveness. Thus, Treg suppress the effector response of virus specific T cells in patients with chronic viral hepatitis infection.
Treg have been shown to exert their suppressive activity through a number of different pathways. Production of immunoregulatory cytokines has been proposed as an important mechanism by which Treg mediate their function. Treg can secrete immune suppressive cytokines including TGF β106 , IL 10 107 and IL 35 108 to limit the functional reactivity of virus specific T cells. Several studies have shown that patients with chronic
15 HCV produce increased amounts of these anti inflammatory mediators. Altogether, Treg activity may play an important role in the maintenance of chronicity and can contribute to immune silencing during chronic HCV infection ( Figure 1 2).
Understanding the actions of the immunoregulatory cytokines that contribute to immune silencing will be critical in developing novel therapeutics to restore immunity towards HCV. Previous work from our laboratory and others have demonstrated that fibrinogen like protein 2 (FGL2) is a novel Treg effector gene, which may influence both the progression to chronicity and the efficacy of antiviral treatment in HCV infection 109
115 .
Figure 1 2. Treg inhibit the adaptive immune response to HCV: Treg control the adaptive immune response to HCV at multiple levels. Through the secretion of anti inflammatory mediators, Treg can inhibit the cytolytic activity of CTL to promote viral persistence. Treg also inhibit helper T cell function and influence the ability to produce neutralizing antibodies. (Figure adapted from Rosen et. al. 2008. Transplantation immunology: what the clinician needs to know for immunotherapy . Gastroenterology;134(6):1789 801 .)
16 I.4 Fibrinogen like protein 2 (FGL2):
FGL2, also known as fibroleukin, was first cloned from CTL and is a member of the fibrinogen related protein superfamily due to its homology (36%) with the β and γ chains of fibrinogen 116 . The fgl2 gene, which has been localized to chromosome 7 and 5 in humans and mice respectively, is composed of two exons that are separated by one intron. The fgl2 gene encodes a protein of 432 amino acids in mice and 439 amino acids in humans. FGL2 in its natural state forms a tetrameric complex 117 119 . Based on sequence and structural analysis, it is predicted that the encoded protein is composed of two major domains, a linear N terminal domain and a globular C terminal domain. The 229 amino acid long carboxyl terminus consists of a conserved globular domain, known as the fibrinogen related domain (FRED) that is characteristic of the fibrinogen related protein superfamily. The overall identity between the mouse and human FGL2 is 78%, but within the FRED domain the two proteins share 90% homology. FGL2 has been shown to exist as both a membrane bound prothrombinase and as a secreted immunosuppressive molecule ( Figure I 3).
17 INNATE IMMUNITY ADAPTIVE IMMUNITY Membrane bound FGL2 Secreted FGL2
Domain 2
Domain 1
• Secreted by Treg Macrophages and • • Immunoregulatory activity endothelial cells •Inhibits DC maturation, • Prothrombinase activity inhibits T cell proliferation and mediated by domain 1 induces B Cell apoptosis
Figure I 3. The contribution of FGL2 to innate and adaptive immunity: FGL2 exists as a membrane bound molecule possessing prothrombinase activity on the surface of reticuloendothelial cells or a molecule possessing immunoregulatory activity that is secreted by Treg (Figure from Shalev et. al. 2010. The Role of FGL2 in the Pathogenesis and Treatment of Hepatitis C Virus Infection. Rambam Maimonides Medical Journal. 1:1 12 .)
I.4.1 Membrane bound FGL2:
In macrophages and endothelial cells, FGL2 is expressed as a type II transmembrane protein possessing prothrombinase activity. The prothrombinase activity of FGL2 is attributable to the serine protease activity at position 89 within its N terminal domain 120 . The prothrombinase activity of FGL2 is dependent on calcium and the interaction with phospholipids and is enhanced by the addition of Factor Va. A number of studies have demonstrated a direct role for the prothrombinase activity of FGL2 in innate immunity, playing critical roles in FVH, xeno and allotransplantation rejection, and fetal loss syndrome 117, 121 144 .
In the context of viral infection specifically, thrombin generated at sites of viral infection, as a result of FGL2 activity can upregulate the expression of a number of
18 chemokines in tissue resident macrophages such as, cytokine induced neutrophil chemoattractant (CINC) 145 146 , monocyte chemoattractant protein 1 (MCP 1) 147 and
IL8 148 . Consequently, thrombin is a potent chemotactic agent that serves to recruit leukocytes to sites of inflammation and reduce viral load at these sites of infection. The activity of FGL2 can further contribute to innate immunity against viral infection through thrombin’s capacity to cleave fibrinogen to fibrin. The deposition of fibrin at sites of viral infection limits viral spread to uninfected neighboring cells by creating a physical barrier surrounding the infected cells 117,129,136 . Consequently, FGL2 controls early viral replication prior to the induction of adaptive immunity.
Lethality in a murine model of mouse hepatitis virus 3 (MHV3) induced FVH is caused by a combination of viral cytopathy and an inappropriate activation of FGL2 prothrombinase activity on reticuloendothlial cells in susceptible strains123,135 136 . In susceptible BALB/C or C57BL/6 mice, the overproduction of thrombin generated through the activation of FGL2 initiates an ischemic hepatocellular necrosis and causes intrasinusoidal thrombi formation. The excessive immunopathology that occurs as a consequence of FGL2 dependent immune coagulation can result in liver failure and ultimately death. A/J mice, which produce less FGL2 in response to MHV3, survive infection by producing an appropriate innate and adaptive immune response, clearing infection by day 15 PI 149 . Fgl2 / mice infected with MHV3 have prolonged survival, when compared to susceptible wild type C57BL/6 mice, but still succumb to infection.
Histological analysis of liver sections from these mice however reveals that the cause of death is due to viral cytopathic effects caused by enhanced viral dissemination, rather than the pathology caused by immune coagulation observed in susceptible strains 150 . In
19 concordance with this, viral titres are increased in surviving fgl2 / mice when compared to resistant A/J mice, highlighting the importance of FGL2 in the early control of MHV3.
I.4.2 Secreted FGL2:
FGL2 is expressed as a secreted protein possessing immunoregulatory activity in
T cells, Treg and other regulatory cell populations such as, CD8 +CD45RC low T cells 151 ,
CD8αα + T cells in the intestine 109 , NKT cells 152 , and CD4 CD8 double negative (DN) T cells 153 . The C terminal globular domain of FGL2 is responsible for its immunosuppressive activity. FGL2 exerts its immunosuppressive activity by binding to
FCγRIIB, expressed differentially on antigen presenting cells (APC) including monocytes/macrophages, DC, B cells, and endothelial cells 154 . In vitro studies have shown that ligation of FGL2 to FCγRIIB prevents the maturation of DC, inhibiting the expression of important maturation markers such as, CD80 and MHC 153 . It was also shown that FGL2 inhibits the secretion of proinflammatory cytokines by inhibiting NFκB translocation into the nucleus 155 .
By inhibiting the maturation of DC, FGL2 indirectly suppresses the activity of T cells. Treatment of splenocytes with FGL2 inhibits T cell proliferative responses towards alloantigens and neutralizing the activity of FGL2 using antibodies restores proliferation 155 . Moreover, FGL2 treatment results in a TH2 polarization in CD4 + T cells with upregulated expression levels of TH2 cytokines, such as IL 4 and IL 10 and downregulated expression levels of TH1 cytokines such as, IL 2 and IFN γ155 . Targeted deletion of fgl2 impairs Treg activity and enhances the reactivity of DC, T and B cells.
Due to the impaired activity of Treg, older fgl2 / mice develop autoimmune glomerulonephritis 154 . The regulatory activity of FGL2 has also been implicated in
20 allograft rejection 154 , cancer 156 158 , autoimmunity 153 , and experimental and human viral infections, including human immunodeficiency virus (HIV)159 , severe acute respiratory syndrome (SARS) 160 , and chronic HBV infection 161 . In an experimental model of FVH caused by MHV3, increased plasma levels of secreted FGL2 as well as increased frequencies of Treg pre and post viral infection are predictive of susceptibility and severity of disease. Inhibition of FGL2 by antibody or siRNA to exon 1 of the mouse fgl2 gene enhances the survival of susceptible animals, whereas adoptive transfer of wild type
Treg into resistant fgl2 deficient ( fgl2 / ) animals accelerates their mortality 162 .
In addition to regulating T cell responses, FGL2 regulates humoral immunity by engaging FCγRIIB to induce the apoptosis of B cells and plasmablasts 154 . In fact, splenocytes isolated from fgl2 / mice have increased numbers of Ab producing B cells in response to stimulation with the T cell independent antigens, LPS or NP Ficoll 153 .
Studying the effect of FGL2 and its relation to HCV persistence may provide insight into the role of FGL2 in inhibiting a robust CTL response to virus infection.
However, given the lack of a small animal model susceptible to infection, a role for FGL2 in promoting HCV persistence can to date only be inferred through a limited number of observational studies in humans. To study the contribution of FGL2 to immune regulation and its effect on the immune response, a well established model of acute viral hepatitis caused by LCMV WE was used 163 .
21 I.5 Lymphocytic Choriomeningitis Virus (LCMV):
LCMV is a single stranded RNA virus that belongs to the Arenaviridae family of viruses 164. The LCMV genome consists of two segments of single stranded RNA – a large (L) segment of approximately 7 kb and a small (S) segment of 3.4 kb 164 169. The L
RNA segment encodes the viral RNA dependent RNA polymerase, whereas the S segment codes for the three major structural proteins: the nucleocapsid protein (NP) and the two surface glycoproteins, GP 1 and GP 2, which are derived through post translational cleavage from a common precursor polypeptide GP C. A well characterized cellular receptor of LCMV is α dystroglycan (α DG), an important cell receptor for ECM proteins 170 176. Encoded as a single protein, DG is cleaved into the extracellular α DG and a membrane anchored β DG. α DG has high affinity interaction with a number of ECM proteins including laminin and percolin. LCMV competes with ECM proteins for binding to α DG. Furthermore, LCMV infection reduces expression of α DG and perturbs DG mediated assembly of ECM, affecting normal cell matrix interactions and functions 170 176.
LCMV has been studied extensively for over a century providing important insights into host virus interaction that has been extended to HIV and HCV infection in humans 166, 177. LCMV has provided researchers with a powerful model to interrogate the immune response to viral pathogens. Some of the seminal studies that have been performed using this model have included a demonstration of MHC I restriction, mechanisms of central tolerance, mechanisms leading to viral persistence and the effect of host genetics on the outcomes of viral infection.
First isolated by Armstrong and Lillie, LCMV produces a fatal acute central nervous system disease when introduced intracranially (IC) in mice and monkeys 178.
22 Several strains have been discovered exhibiting a number of disease manifestation that
are dependent on the route of infection, dose, strain, host genetics and
immunocompetance of the host.
The Effect of Route: Disease Manifestation in Immunocompetent Mice: IC inoculation Fatal lymphocytic choriomeningitis. IV or IP inoculation Systemic infection. Replication and tropism dependant on the strain. Subcutaneous (SC) inoculation Localized swelling of the affected tissue.
*Infection with 2x10 6 PFU IV The Effect of Strain: Disease Manifestation in Immunocompetent Mice: LCMV WE / Aggressive Acute hepatitis cleared 12 14 days post infection LCMV ARM Acute systemic infection cleared 8 10 days post infection LCMV Clone 13 / Docile Chronic Viral Infection
Table 1 2. Route of inoculation and strain of LCMV affect disease manifestation and clearance kinetics.
Infection of mice with LCMV can result in an acute, protracted or lifelong chronic
infection depending on the strain of virus used and the genetic background of the host 179
180. Among the most widely studied LCMV isolates are the Armstrong (ARM) and WE
strains. Their genomes exhibit approximately 84% sequence identity at the nucleotide
level and they share 91% amino acid sequence identity. Moreover, the two strains carry
the same H 2b restricted CTL epitopes. ARM is neurotropic 181 whereas the WE strain is
viscerotropic 182.
I.5.1 LCMV WE:
It is well documented that WE but not ARM causes an acute liver disease in mice,
which resolves by day 14 post infection 163,183 184. The hepatocellular disease and tropism
associated with LCMV WE has been mapped to mutations in the GP gene in segment S
and the polymerase gene in segment L 179 180, as demonstrated by nucleotide
23 fingerprinting 180 and reverse genetic mapping 179. The strengths of studying the contribution of FGL2 to the regulation of immunity in LCMV WE infection are 2 fold: 1)
LCMV is not cytolytic in vivo which allows for a distinct separation of pathological effects caused by the virus (such as direct virus injury and lysis of cells) from those caused by the host’s immune system; and 2) Immunocompetent adult mice that are infected with LCMV WE generate a profound immune response that eliminates the virus.
Several strains of LCMV have been identified and characterized with the ability to induce acute and chronic hepatitis ( Table 1 1). The WE strain of LCMV induces acute viral hepatitis, while LCMV Clone 13 (Cl 13) causes chronic viral infection and T cell exhaustion, and has served as a model to study the pathogenesis of chronic viral infections183, 163, 165.
Table I 3. Comparing the clinical outcomes of infection with different strains of LCMV with chronic HCV infection: LCMV WE, LCMV Cl13 and LCMV Docile have been utilized as models of infection with hepatotropic viruses to study host virus interactions due to their ability to produce acute or chronic liver disease.
Infection of adult mice though an intraperitoneal (IP) or intravenous (IV) route using a high dose of LCMV WE causes an acute hepatitis. Like HCV, liver cell injury
24 and control is dependent on CTL activity within the liver. Additionally, long term control requires the sustained activity of neutralizing antibodies to prevent viral reemergence following resolution. Mice infected with LCMV WE have elevated plasma alanine transaminase (ALT) levels, a marker of liver necrosis following infection. At the peak of infection, there is evidence for panlobular infiltration with necrotic lesions surrounding portal triads. Disease symptoms in this acute model of hepatitis fade parallel to clearance of the virus and the rapid decline of T cell activity within the liver.
I. 6 Hypothesis:
In this study, using a model of acute viral hepatitis caused by LCMV WE, the effect of targeted deletion of fgl2 on the generation of an adaptive immune response to
LCMV was examined. The hypothesis behind these studies is that FGL2 prothrombinase activity generated by reticuloendothelial cells at the site of infection is required to control early viral replication, both through the generation of thrombin at sites of viral infection and the subsequent actions of thrombin creating a fibrin barrier to prevent viral spread early in infection ( Figure I 5).
25
Figure I 5. Proposed model for how FGL2 affects the early phase of viral infection: The prothrombinase activity of FGL2 generates thrombin at sites of viral infection. Thrombin is a powerful chemotactic agent recruiting phagocytes and APC into sites of viral infection. Thrombin also can cleave fibrinogen and promote the formation of a fibrin deposit, limiting viral spread to nearby uninfected cells.
Following the induction of adaptive immunity, it is predicted that secreted FGL2 production by Tregs inhibits virus specific T and B cell immune responses, by engaging the inhibitory FCγRIIB receptor on APC ( Figure I 6). Thus, deletion of fgl2 will result in a restoration of immunity causing an enhancement of DC maturation, increased T cell antiviral responses and increased humoral immunity.
26
Figure I 6. Proposed model for how FGL2 affects the adaptive immune response to viral pathogens: FGL2 produced by Treg cells down regulates DC maturation through binding to the inhibitory Fc γRIIB receptor, resulting in inhibition of T and B cell effector functions. FGL2 can also directly induce apoptosis in B cells upon binding to the inhibitory Fc γRIIB receptor. Figure from Shalev et. al. 2008. Targeted Deletion of fgl2 Leads to Impaired Regulatory T Cell Activity and Development of Autoimmune Glomerulonephritis. J Immunol.180:249 260.
27 CHAPTER II
Materials and Methods
II.1 Mice:
Fgl2 / mice were generated as previously described 150 . Fgl2 / mice were maintained on a C57BL/6 background. Female fgl2 / mice and wild type C57BL/6 littermate controls weighing 20 25 grams were maintained in micro isolator cages and housed under specific pathogen free (SPF) conditions in the animal colony at the Princess
Margaret Hospital, University of Toronto. Mice were fed a standard lab chow diet and water. All animal experiments in the study were carried out according to the guidelines set by the Canadian Council on Animal Care. Protocols were approved by members of
Animal Care Committee at the University Health Network.
II.2 Virus:
LCMV WE was obtained as a gift from the laboratory of Dr. Pamela Ohashi
(Ontario Cancer Institute, Toronto, ON) and was propagated on L929 cells (ATCC
#CCL 1). LCMV Cl 13 was obtained as a gift from the laboratory of Dr. Michael
Oldstone (The Scripps Research Institute, La Jolla, CA) and was propagated on BHK 21 cells (ATCC #CCL 10). Virus was purified from virus containing supernatants by banding on isopycnic Renografin 76 (Sigma Aldrich, St. Louis, MO) gradients as described previously 185 . Purified virus was diluted to 2 mg/mL in TNE buffer and stored at 80ºC until use. Virus titres were measured using a focus forming assay as described
186 .Weight matched female fgl2 / mice and wild type C57BL/6 littermate controls were infected IV with 2x10 6 PFU of LCMV WE or LCMV Cl 13. To test immunological
28 memory and protection, mice were rechallenged intravenously with 2x10 6 PFU of LCMV
WE.
II.3 Cell Culture Media:
All media for cell culture was purchased from the Ontario Cancer Institute (OCI)
(Princess Margaret Hospital, Toronto, ON).
II.4 Synthetic Peptides:
b b The LCMV peptides, GP 33 41 H2 D (KAVYNFATC), NP 396 404 H2 D
b (FQPQNGQFI) and GP 61 80 I A (GLNGPDIYKGVYQFKSVEFD) were synthesized by
Anaspec, Inc. (San Jose, CA) with a purity of >95%.
II.5 Blood Collection:
Blood was collected by saphenous venipuncture or heart puncture using heparinized capillary blood collection tubes (Fisher Scientific Co., Toronto, ON). Blood was centrifuged and plasma was collected. Plasma aliquots were made and stored at
80ºC until use.
II.6 Measurement of ALT:
10 L of plasma was transferred onto a Vitros II ALT slide and analyzed using a
Vitros DT60 II Chemistry System (Ortho Clinical Diagnostics, Neckargemünd,
Germany).
II.7 Measurement of Plasma FGL2:
Monoclonal mouse anti mouse FGL2 (clone 6H12) and polyclonal rabbit anti mouse FGL2 were produced as previously described 154 155 . Recombinant poly histadine tagged mouse FGL2 was expressed in a mammalian Chinese Hamster Ovary (CHO) cell system as previously described 155 . Recombinant FGL2 was purified using a Ni NTA
29 Purification System (Invitrogen, Carlsbad, CA). The purity of recombinant FGL2 was confirmed by SDS–PAGE and Western blot analysis as previously described 154 155 .
A sandwich enzyme linked immunosorbant assay (ELISA) for the measurement of FGL2 in plasma has been developed in our laboratory 152 . Briefly, certified high binding 96 well plates (Costar EIA/RIA, Corning Inc., Corning, NY) were coated with 50 ng / well of monoclonal anti mouse FGL2 (monoclonal IgG1 6H12) overnight at 4ºC.
After washing in 0.05 % Tween 20 / Tris buffered saline (TTBS), non specific binding of
FGL2 to the plates was reduced by incubating plates with 230 L / well of SuperBlock solution (Pierce Biotechnology Inc., Rockford, IL) for 1 hour. Recombinant mouse FGL2 was serially diluted in 2.5 % bovine serum albumin (BSA) in phosphate buffered saline
(PBS) to create a standard curve. 50 L / well of either, recombinant mouse FGL2 or mouse plasma (diluted 1:6 with 2.5% BSA in PBS) was subsequently added to the plates and allowed to adhere for 1 hour. After washing with TTBS, the plates were incubated with 50 ng / well of a polyclonal rabbit anti mouse FGL2 antibody diluted with 1% BSA in PBS. The plates were washed with TTBS and 50 L / well of goat anti rabbit antibody conjugated to horseradish peroxidase (HRP) (diluted at 1:6000 with 1% BSA in PBS) was added to the plates and incubated for 1 hour. The plates were washed with TTBS and 100 L / well of tetramethyl benzidine (TMB) (Sigma Aldrich, Oakville, ON) was added to the plates. The reaction was terminated after 5 minutes with the addition 100 l
/ well of 1M H 2(SO 4). The absorbance value at 450 nm was measured using an Appliskan multimode microplate reader (Thermo Fisher Scientific Inc., Waltham, MA). The absorbance value measured at 450 nm correlated with the amount of FGL2 within each well and detected levels as low as 100 pg per sample.
30 II.8 Isolation of Lymphocytes from Spleens and Lymph odes:
Spleens and inguinal, axillary and brachial lymph nodes were isolated in Hanks
Balanced Salt Solution (HBSS) and filtered through a 40 m nylon mesh. Cells were treated with RBC lysis buffer (Ebioscience, San Diego, CA) for 5 minutes on ice and washed before further processing. To assess the maturation status of DC, spleens and lymph nodes were isolated and minced in a petri dish containing serum free RPMI 1640 with 1 mg / mL Collagenase D (Roche Applied Science, Indianapolis, IN) and 0.02 mg/mL DNAse I. The tissue was digested at 37ºC for 40 minutes and was subsequently filtered using a 70 um nylon mesh. The maturation status of DC was assessed by examining the median fluorescence intensity (MFI) following staining of the markers,
CD80 and MHCII by flow cytometry.
II.9 Isolation of Intrahepatic Mononuclear Cells:
Livers were infused through the inferior vena cava with digestion media (serum free RPMI 1640 containing 0.2 mg/mL Collagenase IV (Sigma Aldrich, St. Louis, MO) and 0.02 mg/mL DNAse I (Roche Applied Science, Indianapolis, IN)) at a rate of 7 mL/minute. Livers were minced and transferred to petri dishes containing digestion media and incubated for 40 minutes in a 37ºC waterbath. The reaction was terminated by the addition of iced cold serum free RPMI 1640 containing 1mM
Ethylenediaminetetraacetic acid (EDTA) (Sigma Aldrich, St. Louis, MO). Liver tissue was filtered using a 100 m nylon mesh and subsequently centrifuged at 30 g for 3 minutes. The supernatant containing nonparenchymal cells was collected and the cells were washed 2 times. Liver nonparenchymal mononuclear cells were isolated by density centrifugation on Lympholyte M (Cedarlane Laboratories, Toronto, ON).
31 II.10 Histology and Immunohistochemistry:
Liver tissue was embedded in paraffin by immersion of the median lobe of the liver in 10 % formalin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 48 hours, while shaking. Formalin fixed tissue was submitted to the Pathology Core Facility at Toronto General Hospital. Tissue was embedded in paraffin, sectioned and stained with H&E.
For immunohistochemistry, frozen sections were prepared by embedding the right lobe of the liver in Optimal Cutting Temperature Compound (Tissue Tek, Sakura
Finetek, Torrence, CA) filled cryomolds. Cryomolds were frozen in liquid nitrogen and processed at the Pathology Core Facility at Toronto General Hospital. Tissue was cut into
5 m thick sections and stained using a rat anti mouse CD8α antibody (Clone 53 6.7; eBioscience, San Diego, California, USA) or a rat anti LCMV NP antibody (VL4 Ascites
(1:50 dilution)). Tissue was incubated with a secondary anti rat antibody conjugated with
HRP that stained positive cells brown after addition of the substrate 3,3’ diaminobenzidine (DAB) (Zymed, San Francisco, CA).
II.11 Morphometric Analysis:
Slides were digitally scanned and the number of CD8 + cells was quantified by morphometric analysis using Spectrum V.10.2.2.2317 (Aperio Technologies Inc., Vista,
California, USA). The number of CD8 + cells was expressed as a percent of total infiltrating mononuclear cells.
II.12 Chromozym Th assay:
A peritoneal lavage was performed on day 3 thioglycolate primed fgl2 / or wild type mice using HBSS. The adherent cell population was found to be ≥ 80% F4/80 +
32 macrophages by flow cytometry. 3x10 5 macrophages were seeded onto flat bottom tissue culture treated 96 well plates and allowed to adhere to the plate for 4 hours at 37ºC / 5 %
CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum
(FCS), L glutamine (L Glut) and antibiotics (penicillin and streptomycin). Following adherence to plastic, the medium was replaced with DMEM supplemented with 2 % FCS/
L Glut / antibiotics containing LCMV WE at the indicated multiplicity of infection
(MOI) for 1 hour. The infectious innoculum was removed and DMEM containing 10 %
FCS / L Glut / antibiotics was added. Following 2 days of infection, 500 ng of murine prothrombin (Genway Biotech, San Diego, CA, USA) was applied to the cells in 20 uL for 20 minutes at 37ºC / 5 % CO 2. 125 uL of iced cold assay buffer (50mM Tris, 227 mM
NaCl, 1% BSA, pH 8.3) was added after to terminate the prothrombinase reaction.
Supernatants were assessed for thrombin activity by the addition of 15 uL of the chromogenic thrombin substrate, Chromozym Th (Roche Applied Science, Indianapolis,
IN). The absorbance value was measured at 420 nm every 10 minutes for 4 hours. The rate of absorbance change (( Abs@420nm) / min) correlated with prothrombinase activity within each well.
II.13 In Vitro Infection of Fibroblasts with LCMV in the Presence of FGL2:
2x10 5 MC57 fibroblasts were seeded onto 24 well plates. Following adherence to plates, cells were treated with increasing doses of recombinant his tagged FGL2 for 1 hour. Cells were subsequently infected with 0.01 MOI LCMV WE for 1 hour. The infectious innoculum was removed and DMEM containing 10 % FCS /L glut/antibiotics was added to the cells. The viral titre of the supernatant was assessed by an LCMV focus forming assay.
33 II.14 Flow Cytometry:
Antibodies used for Flow Cytometry: The following antibodies were used to stain cells for FACS analysis: PE Cy5.5 CD3ε (Clone 17A2), PE CD4 (Clone GK1.5), PE
CD8α(Clone 53 6.7), APC CD80 (Clone 16 10A1), FITC MHCII (I A) (Clone NIMR
4), FITC IFN γ (Clone XMG1.2), CD16/ CD32 (Clone 93), PE CD11c (Clone N418).
Tetramers used for Flow Cytometry: The following MHC tetramers were used to evaluate the antigen specificity of T cells:
b MHC I:ALEXA FLUOR® 647 GP 33 41 H2 D and ALEXA FLUOR® 647 P 396 404 H2
Db
Tetramers were provided by the NIH Tetramer Core Facility (Emory University Vaccine
Center, Atlanta, GA).
Cell staining: Mononuclear cells were isolated from spleen, lymph nodes (inguinal, axillary and brachial) and livers were washed and suspended in FACS buffer (PBS
7 containing 1% FCS and 1 mM EDTA) at a final concentration of 1x10 cells/ml. Cells were treated with antibodies towards CD16/CD32 to block non specific binding to FC receptors. Cells were surface stained with antibodies and LCMV specific tetramers.
Cells were fixed with 2 % paraformaldehyde. FACS analysis was conducted using a BD
LSRII Flow Cytometer and data were analyzed using FlowJo software (Tree Star, Inc.,
Ashland, OR). Live cells were discriminated according to forward scatter and side scatter parameters or using a fixable viability dye (Ebioscience, San Diego, CA).
II.15 Assessing LCMV Specific Humoral Responses:
An LCMV ELISA for the detection of total LCMV specific antibodies has been previously described 187 . Briefly, certified high binding 96 well plates were coated for 24
34 hours at 4 ºC with 100 ng/well of purified LCMV WE that was diluted in carbonate bicarbonate buffer (1.59 g Na 2CO 3, 2.93 g NaHCO 3 in 1 L ddH 2O, pH 9.6 ). Plates were washed with TTBS and non specific binding of antibody to the plates was reduced by incubating plates with 230 L SuperBlock solution (Pierce Biotechnology Inc., Rockford,
IL) for 1 hour. Plasma samples were serially diluted in 2.5 % BSA in PBS and were transferred to the plates and incubated for 1 hour at 37 ºC. Plates were washed with TTBS and an HRP conjugated goat anti mouse IgG1 antibody used at a 1:6000 dilution in 1 %
BSA/PBS was added to each well. The plates were incubated at 37 ºC for 1 hour. Plates were washed with TTBS and 100 L / well of TMB was added to the plate. The reaction was halted after 5 minutes by the addition of 100 L / well of 1M H 2(SO 4). The optical density reading at 450 nm was measured using an Appliskan multimode microplate reader (Thermo Fisher Scientific Inc., Waltham, MA). The absorbance value measured at
450 nm correlated with the captured total LCMV specific antibody within plasma samples. The dilution series for each plasma sample was plotted and read where the dilution and observed absorbance values had a linear relationship with one another.
Samples were expressed as a fold increase from naïve absorbance.
II.16 Measurment of eutralizing antibodies:
LCMV neutralizing antibody titres were quantified in mouse plasma by a plaque reduction assay 188 . Briefly, mouse plasma from infected or naïve mice was serially diluted and incubated with 200 PFU of LCMV WE for 1 hour. Following incubation, a standard focus forming assay was performed to visualize the number of plaques of
LCMV formed. The neutralizing antibody titre was defined as the dilution that
35 neutralized 50 % of the plaques formed by the incubating 200 PFU of virus with control plasma from uninfected mice.
II.17 Intracellular Cytokine Analysis:
Lymphocytes were stimulated with 1 ug of MHC class I restricted LCMV GP 33 41 or NP 396 404 peptide or MHC class II restricted LCMV GP 61 80 for 6 hours. 10 g/mL of
Brefeldin A (Sigma Aldrich, St. Louis, MO) was added to cultures to block secretion of
IFN γ. Cells were stained for surface expression of CD4 or CD8α. Cells were fixed with
2 % paraformaldehyde, permeabilized with 1% Saponin (Sigma Aldrich, St. Louis, MO) in FACS buffer and stained with an antibody to IFN γ. Antibodies to CD16/ CD32 were used to block FC mediated binding to FC receptors. Intracellular expression of IFN γ was assessed by flow cytometric analysis using a Digital LSR II (Becton Dickinson).
II.18 Statistical analysis:
Results were reported as the mean and standard error of mean (SEM) unless otherwise specified. An ANOVA was performed to examine differences between groups.
Differences with P ≤ 0.05 were considered significant.
36 CHAPTER III
RESULTS
III.1 The Prothrombinase Activity of FGL2 Prevents Early Viral
Dissemination in Wild Type Mice:
To investigate the effect of LCMV WE infection on the induction of the prothrombinase activity of FGL2, peritoneal macrophages were isolated from day 3 thioglycolate primed wild type or fgl2 / mice and subsequently infected in vitro with
LCMV WE at different MOI. Following 2 days of infection, the prothrombinase activity of infected macrophages was assessed by measuring thrombin generation after incubation with prothrombin using a colorimetric Chromozym Th assay. The rate of cleavage of the
Chromozym Th substrate has been shown to correlate with thrombin generation 122 . In macrophages isolated from wild type mice, thrombin was generated in a dose dependent manner with increasing MOI of LCMV. Following LCMV WE infection, no induction of prothrombinase activity was seen in macrophages isolated from fgl2 / mice ( Figure III
1.1A ). To evaluate whether the observed prothrombinase activity limits viral dissemination in vivo , livers were recovered from wild type and fgl2 / mice at various time points PI with LCMV WE and viral titres were measured using an LCMV focus forming assay ( Figure III 1.2A ). Livers were also immunostained for the NP of LCMV
(Figure III 1.2B) . Tissue isolated from naïve uninfected wild type and fgl2 / mice was negative for virus in the LCMV focus forming assay and immunohistochemical staining for the NP of LCMV (data not shown). Following infection however, the loss of fgl2 impaired innate viral control and resulted in enhanced viral replication early in infection.
37 1.60E 03 * 1.40E 03 1.20E 03 * 1.00E 03 8.00E 04 * 6.00E 04 ODnm) (420 min / 4.00E 04 OD(420nm)/min 2.00E 04
0.00E+00 0.01 0.001 0
MultiplicityMultiplicity of Infection of Infection
Figure III 1.1. FGL2 prothrombinase activity is induced in macrophages upon LCMV infection: Peritoneal exudate macrophages (PEM) were isolated from the day 3 thioglycolate primed C57BL/6J and fgl2 / mice and subsequently infected in vitro with LCMV WE at different MOI. The prothrombinase activity of FGL2 was assessed in a chromogenic assay utilizing Chromozym Th as a substrate. Data shown as the mean rate of cleavage of chromozym Th ± SEM and is representative of 3 independent experiments with 5 mice per group. A B
Figure III 1.2. Fgl2 / mice have enhanced viral replication prior to the induction of adaptive immunity: Fgl2 / and wild type mice were infected with 2x10 6 PFU of LCMV WE. An LCMV focus forming assay (A) and immunohistochemical staining for the NP of LCMV (B) was utilized to detect viral titre within livers. Liver tissue was taken from 5 mice at the indicated time points post infection. Representative images are shown for immunohistochemical staining.
38 By day 4 PI with LCMV WE, viral titres were increased 100 fold in fgl2 / mice. The observed enhanced viral replication was evident until day 6 PI, coinciding with the induction of adaptive immunity. These findings are consistent with the loss of prothrombinase activity of FGL2 in fgl2 / mice, which has been shown to limit replication and viral spread 150 .
To evaluate the contribution of secreted FGL2 on the observed protective effect of membrane bound FGL2 in early viral control and LCMV WE replication, MC57 fibroblasts with no prothrombinase activity of their own (data not shown) were treated with increasing doses of a mutated recombinant FGL2 protein also devoid of prothrombinase activity 120 . These cells were subsequently infected with LCMV WE at an
MOI of 0.01 to assess whether secreted FGL2 limited the production of LCMV.
Figure III 1.3. FGL2 does not mediate its protective effects by inhibiting viral entry or production: MC57 cells were incubated with increasing doses of recombinant FGL2 for 1 hour and subsequently infected with LCMV WE at an MOI of 0.01. Viral titres were measured within the supernatant at 18 hours, 24 hours and 48 hours.
39 The viability of MC57 fibroblasts was not significantly altered by the presence of FGL2 for 48 hours of culture (data not shown). Viral titres were measured at various time points early after infection. It was observed that secreted FGL2 did not significantly inhibit the production of LCMV WE in MC57 up until 48 hours post infection ( Figure
III 1.3 ).
III.2 Secreted FGL2 is Induced Following LCMV Infection of Wild Type
Mice:
Secreted FGL2 was measured in vivo by infecting wild type or fgl2 / mice with
2x10 6 PFU of LCMV WE IV. Mice were bled at various time points PI by saphenous venipuncture. FGL2 levels were monitored within the plasma of infected mice using an
FGL2 sandwich ELISA. The concentration of FGL2 in plasma from naïve wild type mice was measured to be 0.8 ± 0.2 ng/mL, while FGL2 was undetectable in fgl2 / mice both pre and post infection at all time points (data not shown). As early as 2 days PI, FGL2 levels increased significantly reaching a peak expression at day 8 PI (7.8 ± 0.5 ng/mL).
FGL2 levels remained significantly elevated when compared to naïve values following viral clearance from the liver (day 12) until 40 days PI ( Figure III 2.1 ).
40 Figure III 2.1. FGL2 is induced in vivo upon LCMV WE infection and remains elevated long after viral clearance from the liver: C57BL/6 mice were bled at various time points following infection with 2x10 6 PFU of LCMV WE. FGL2 was measured in plasma by ELISA. Values shown are the mean FGL2 concentrations of 5 mice per time point and are expressed as mean ± SEM.
III.3 Targeted Deletion of fgl2 Results in Enhanced Intrahepatic Antiviral
Activity Following LCMV Infection:
To evaluate antiviral inflammatory activity within the liver during the course of infection, histological analysis of liver cross sections stained with H&E was performed.
Livers from naïve fgl2 / mice revealed that, in the absence of infection, the livers appeared histologically normal ( Figure III 3.1A.Day aïve ). From days 4 to 6 PI, there was evidence for periportal lymphocytic infiltration in both mice. However, increased numbers of mononuclear cells were observed within the infiltrate in fgl2 / mouse livers
(Figure III 3.1A.Day 4 6). By day 8 PI, panlobular lymphocytic infiltration was observed in both mice. However, fgl2 / mice had increased lymphocytic infiltrates and hepatic necrosis, which was only minimally evident in wild type mice ( Figure III
41 3.1A.Day 8 ). By day 10 PI, livers isolated from fgl2 / mice showed large necrotic lesions and bridging necrosis, while the architecture of the livers isolated from wild type mice were preserved ( Figure III 3.1A.Day 10 ). As the infection resolved at day 12 PI, both wild type and fgl2 / mice recovered significantly ( Figure III 3.1A.Day 12 ). To validate the histological data, ALT, a biomarker of liver necrosis and liver inflammation, was measured in the plasma of virally infected wild type or fgl2 / mice. Naïve wild type and fgl2 / mice had similar plasma ALT levels prior to infection (18.7 ± 2.9 vs. 19.2 ± 1.4
IU/L respectively ). After infection, ALT levels increased significantly in both wild type and fgl2 / mice. In support of the histological finding that there was enhanced antiviral inflammation in fgl2 / livers, fgl2 / mice had increased expression of plasma ALT when compared to wild type mice at all time points measured ( Figure III 3.1B ).
42 A Wild -Type Fgl2 -/- B ALT Levels in Plasma Post Infection with 2x10^6 PFU of Naive ALTLCMV Levels WE
3500 * * 3000 2500
2000 * Wild Type 1500 FGL2 Knockout
Day 4 (IU/L) ALT 1000
500
0 Day 8 Day 10 Day 12
Day 6
Figure III 3.1. Targeted deletion of fgl2 results in enhanced inflammatory responses within the liver towards LCMV infection: Wild type or fgl2 / were infected with 2x10 6 PFU of LCMV WE. Day 8 To assess liver histopathology, livers were isolated at various time points post infection and were subsequently sectioned and stained with H&E. Representative photographs of 3 livers were taken to illustrate the liver pathology that is observed during the course of infection (A) . ALT Day 10 levels were measured to quantify the extent of liver necrosis as a result of antiviral activity within the liver (B) . Values shown are the mean ± SEM of 4 mice per time point.
Day 12
43 Correlating with the enhanced inflammation observed in fgl2 / livers, there was a reduction in viral titres to levels comparable to that of wild type mice following the induction of adaptive immunity. Thus, despite the innate defect in viral containment observed on day 4 of infection, fgl2 / mice mounted a strong adaptive immune response that results in similar clearance kinetics as wild type mice. ( Figure III 3.2 ).
Figure III 3.2. Fgl2 / mice and wild type mice have similar viral clearance kinetics, despite enhanced early viral replication in fgl2 / mice: Both fgl2 / and wild type mice were infected with 2x10 6 PFU of LCMV WE. Liver tissue was taken from 5 mice at various time points post infection. An LCMV focus forming assay was utilized to detect virus within livers.
44 III.4 Dendritic Cells Isolated From fgl2 / Mice Have Increased Activity
Following Infection with LCMV WE:
To assess the maturation status of DC following infection, both wild type and fgl2 / mice were infected with 2x10 6 PFU of LCMV WE. After 1 day PI, splenocytes were isolated and subject to flow cytometric analysis. The activation of CD11c + DC was determined by examining the MFI of the activation markers, CD80 and MHCII. It was found that at day 1 PI (Figure III 4.1), the expression levels of CD80 and MHCII were enhanced on splenic DC isolated from fgl2 / mice.
CD80 MHCII
700 * 7000 *
600 6000
500 5000
400 4000
300 3000
200 2000 MedianIntensity Intensity FluorescenceFluorescence 100 intensity median fluorescence 1000
0 Median Fluorescence Intensity Fluorescence Median Median Fluorescence Intensity Fluorescence Median 0 Wild Type FGL2 Knockout Wild type FGL2 Knockout Wild typeWild TypeFGL2 fgl2 Knockout Knockout
Figure III 4.1. Fgl2 / mice have enhanced DC activation at day 1 following infection with LCMV WE: Lymphocytes were harvested from the spleen and subject to flow cytometric analysis of CD11c + DCs for expression of CD80 and MHCII to assess the activation status of dendritic cells. Expression of CD80 and MHCII is displayed as the mean MFI of 3 mice per group ± SEM.
45 III.5 Targeted Deletion of fgl2 Enhances the Frequency of Virus Specific T
Cells and Increases Responsiveness to Ex Vivo Peptide Restimulation Following
LCMV WE:
To assess if enhanced DC activation following infection had the functional consequence of enhancing the antiviral CD8 + T cell response within fgl2 / mice, liver sections from infected wild type or fgl2 / mice were immunostained for CD8 and quantified using a morphometric analysis software.
Figure III 5.1. Fgl2 / mice have increased frequencies of intrahepatic virus specific CD8 + T cells following infection with LCMV WE: Wild type and fgl2 / mice were infected with 2x10 6 PFU of LCMV WE. Liver mononuclear cells were isolated at day 8 PI and stained with MHC tetramers, GP 33 41 H2 b b D and NP 396 404 H2 D (A B). Flow Plots are representative of 4 different mice per group. Values are expressed as the mean % CD3ε+CD8α +Tetramer + of total CD3ε+ CD8α + ± SEM. Liver sections were stained for CD8α+ and morphometry was utilized to assess the mean % CD8 of total infiltrating mononuclear cells ± SEM (C) of 6 mice per group.
46 By day 6 PI, LCMV infection caused a significant increase in the number of CD8 + cells within the livers of both fgl2 / and wild type mice from naïve levels values (data not shown). At day 8 PI, the frequency of CD8 + T cells was significantly greater in the fgl2 / mice compared with wild type mice (Figure III 5.1 (C)). To corroborate this data, liver mononuclear cells were isolated from wild type or fgl2 / mice day 8 PI with LCMV WE
b b and were stained with the class MHC I tetramers, GP33 41 H2 D and NP 396 404 H2 D .
Significantly increased frequencies of intrahepatic virus specific CTL were seen in fgl2 / mice ( Figure III 5.1 (A B) )
To examine whether or not the increased numbers of virus specific T cells in fgl2 / mice were functional, lymphocytes were harvested at various time points PI and were subjected to ex vivo peptide restimulation. If a T cell is functional and possesses a
TCR that is specific to the stimulating peptide, the cell will produce pro inflammatory cytokines such as, IFN γ. CTL responses to peptide were measured by restimulating the harvested lymphocytes ex vivo with the LCMV derived immunodominant MHC I restricted peptides, GP 33 41 and NP 396 404 , while CD4 responses were measured by stimulating lymphocytes with the LCMV derived immunodominant MHC II restricted peptide, GP 61 80 . Following restimulation, flow cytometric analysis was utilized to quantify the frequency of CD8 +IFN γ+ or CD4 +IFN γ+ cells. As expected, very few lymphocytes isolated from either naïve wild type and naïve fgl2 / mice produced IFN γ to in response to GP 33 41 , NP 396 404 or GP 61 80 stimulation. Stimulation of lymphocytes from infected wild type and fgl2 / mice with LCMV derived peptides resulted in an increased frequency of cells secreting IFN γ when compared to the no peptide control. At day 8 PI, there were greater frequencies of cells responding to GP 33 41 or NP 396 404
47 peptide restimulation in fgl2 / mice when compared to wild type with increased frequencies of CD8 +IFN γ+ / TOTAL CD8 + and absolute numbers of CD8 +IFN γ+ cells
(data not shown) responding to peptide restimulation in fgl2 / mice (Figure III
5.2 ).Furthermore, there were enhanced frequencies of CD4 +IFN γ+ cells responding to
/ GP 61 80 peptide restimulation isolated from fgl2 mice at day 8 PI, indicating that in addition to an enhanced CD8 response, there was also an increased CD4 response
(Figure III 5.3 ). Consistent with findings of others, there were increased frequencies of
IFN γ+ T cells following acute infection, when compared to uninfected controls in the absence of peptide stimulation. This observed effect is due to harvesting cells during acute infection when stimulation by LCMV and expansion of CD8 + IFN γ+ T cells had occurred in vivo (no peptide stimulation). Consequently, because lymphocytes are harvested during the active phase of infection, a no peptide control was used to assess baseline activation as a consequence of infection.
48 Naïve Day 8 Wild Type FGL2 Knockout Wild Type FGL2 Knockout 4 4 10 10 1.67 3.47 0.0336 0.146 105 105 103 103 104 104 GP33 102 102 103 103 1 1
FL1 H: IFNgamma IFNgamma 10 FL1 H: IFNgamma IFNgamma 10
3 3 10 10 104 104 NP396 102 102 103 103
1 1
FL1 H: IFNgamma 10 FL1 H: IFNgamma 10 gamma IFN
3 4 4 10 103 10 10
2 No Peptide 10 102 103 103
1 1 FL1 H: IFNgamma 10 FL1 H: IFNgamma 10
0 0 IFN γ 10 10 3 4 5 3 4 5 100 101 102 103 104 0 1 2 3 4 0 10 10 10 0 10 10 10 10 10 10 10 10
CD8 5
* 4
+/ Cells SEM + 3 *
/ Total CD8 Total/ 2 +
IFNγ + Cells %CD8+IFNG+/Total CD8+ 1
% CD8 0 GP33GP33NP396 NP396NO NO PEPTIDEPEPTIDE
Figure III 5.2. Fgl2 / mice have increased frequencies of CD8 + T cells responding to peptide restimulation following LCMV infection Splenocytes were isolated from the spleen of wild type or fgl2 / mice on day 8 following infection with 2x10 6 PFU of LCMV WE. To assess the CD8 response, cells were re stimulated in culture for 6 hours in the presence of class I peptides GP33 and NP396 or with no peptide stimulation. Brefeldin A was added to the cell cultures to prevent cytokine secretion and the percentage of IFN γ+ of total CD8 + T cells was assessed by flow cytometry analysis. Representative flow plots are displayed and graphs show the mean %CD8/Total CD8 ± SEM of 6 mice per group.
49 Naïve Day 8 Wild Type FGL2 Knockout Wild Type FGL2 Knockout 5 0.694 5 1.81 105 0.181 105 0.168 10 10
4 4 104 104 10 10
GP61 103 103 103 103
10 10 gamma
3 4 5 3 4 5 0 102 103 104 105 0 102 103 104 105 0 10 10 10 0 10 10 10
0.398 0.268 105 0.128 105 0.12 105 105
4 4 No Peptide 10 10 104 104
3 3 10 10 103 103
102 102
3 CD4
*
Cells +/ Cells SEM 2 + +
/CD4 Total
+ 1
%CD4+IFNG+/Total CD4+ %CD4+IFNG+/TotalCells CD4+ IFNγ +
%CD4 0 GP61GP61NO NO PEPTIDE PEPTIDE
Figure III 5.3. Fgl2 / mice have increased frequencies of CD4 + T cells responding to peptide restimulation following LCMV infection: Mononuclear cells were isolated from the spleen of wild type or fgl2 / mice on day 8 following infection with 2x10 6 PFU of LCMV WE. To assess the CD4 response, cells were re stimulated in culture for 6 hours in the presence of class II peptide GP61 or with no peptide stimulation. Brefeldin A was added to the cell cultures to prevent cytokine secretion and the percentage of IFN γ+ of total CD4 + T cells was assessed by flow cytometry analysis. Graphs show the mean ± SEM of 5 mice per group.
50 III.6 Targeted Deletion of FGL2 Enhances Humoral Responses Towards LCMV:
Fgl2 / mice and wild type mice were infected with 2x10 6 PFU of LCMV WE and the humoral response was assessed by evaluating the numbers of
CD138 +CD19 low CD45Rlow plasma cells, the titre of total virus specific antibody and neutralizing antibody. The numbers of plasma cells prior to infection were equivalent in both wild type and fgl2 / mice. At day 8 PI, the number of plasma cells significantly increased in both mice. Nevertheless, fgl2 / mice had approximately ~1.5 times the numbers of plasma cells within the spleen ( Figure III 6.1A ). Total LCMV specific IgG antibody was measured in plasma by ELISA. Antibody responses were greater at all time points measured and peaked earlier at week 3 PI in fgl2 / mice when compared with wild type mice. By week 4 PI, the total LCMV specific IgG antibody response had subsided, but remained elevated over wild type antibody titres over 6 weeks PI ( Figure
III 6.1B ). Neutralizing antibody titres were delayed both in wild type and fgl2 / mice, appearing at week 8 PI (data not shown). Consistent with the literature, the production of neutralizing antibodies was severely blunted at all the time points measured in wild type mice. However, fgl2 / mice developed clinically significant titres of neutralizing antibodies towards LCMV WE, peaking at week 16 PI (Figure III 6.1C ).
51 AB ) Low 1.80E+05 * 12000 *
CD45R 1.60E+05
Low 10000 1.40E+05 CD19 + 1.20E+05 8000
1.00E+05 6000 * * * 8.00E+04
6.00E+04 4000
4.00E+04 Fold Induction Naive Over 2000 * 2.00E+04 * 0.00E+00 0 Number of Plasma Cells (CD138 Number of Plasma Cells Day 0 Day 8 Day 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Day 0 Day 8 C
120
100
80 *
60 Plaques 40 * *
20
0 Dilution of Plasma Required to Neutralize 50 to 50 of% ofNeutralize Dilution Required Plasma Week 12 Week 14 Week 16 Week 18
Figure III 6.1. Fgl2 / mice have increased humoral responses towards LCMV: Fgl2 / mice and wild type mice were infected with 2x10 6 PFU of LCMV WE and the humoral response was assessed. CD138 +CD19 low CD45R low plasma cell numbers were evaluated by flow cytometry ( A). Total LCMV specific IgG antibody titres were evaluated by ELISA ( B). UV inactivated Renograffin 76 purified LCMV was adsorbed to a 96 well plate and serially diluted plasma was added to the 96 well plate. Captured antibody was detected using an HRP conjugated goat anti mouse IgG antibody and subsequently developed with TMB. Absorbance values were read in the linear portion of the dilution series. Values shown are expressed as the (absorbance value of sample @450 nm x dilution) / (absorbance value of naïve plasma @450 nm). Neutralizing antibody titres were determine using a standard plaque reduction and are expressed as the dilution of plasma that reduces 200 PFU of virus by 50 % ( C). All values shown are displayed as mean ± SEM and are representative of 4 mice per group.
52 III.7 Targeted Deletion of fgl2 Enhances Immunity to LCMV Upon in Secondary
Reinfection:
To understand how FGL2 regulates both the humoral and the cellular response in a secondary rechallenge, mice were infected with 2x10 6 PFU of LCMV WE for 120 days and then reinfected with the same dose. At day 5 and 10 post reinfection, plasma ALT was assessed to evaluate the degree of protection that neutralizing antibodies developed within the primary response had conferred on mice reinfected with LCMV. Both histological examinations of liver sections following reinfection and biochemical analysis revealed a significant degree of protection from reinfection in both mice when compared to the primary response. However, histological analysis of liver sections from wild type mice showed areas of mild periportal infiltration at day 10 post reinfection (Figure III
7.1A ) and elevated ALT at day 5 post reinfection ( Figure III 7.1B), whereas fgl2 / livers were histologically normal with normal levels of ALT. T cell responsiveness to peptide was also evaluated at day 10 post reinfection. As in the primary response, the frequencies of CD8 + T cells responding to peptide restimulation increased upon reinfection (data not shown). Fgl2 / mice produced a more vigorous response to reinfection with LCMV WE
(Figure III 7.2) with increased frequencies of cells responding to ex vivo peptide restimulation both at 10 post reinfection .
53
Figure III 7.1 Fgl2 -/- mice are completely protected in secondary reinfection with LCMV WE: Fgl2 / mice or wild type mice were infected with 2x10 6 PFU of LCMV WE. 120 days PI mice were reinfected with the same dose. At 5 and 10 days post reinfection, blood was taken by saphenous venupuncture and ALT was evaluated. ALT values are expressed as mean ± SEM. Liver samples were taken from mice at day 10 post reinfection and they were sectioned and stained with using a trichrome stain (i) and H&E (ii). Representative images were taken from 4 mice per group.
54 WILD TYPE FGL2 KNOCKOUT 5 7.87 5 12.7 10 10 10 4 10 4
GP33 3 3 10 10
10 2 10 2
5 2.56 5 3.65 10 10 12 10 10 4 10 4 NP396 8 3 3 10 10 6 * 10 2 10 2
10 4 10 4 NO PEPTIDE 3 3 10 10
10 2 10 2
IFN γ 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Figure III 7.2. Fgl2 / mice have increased frequencies of CD8 + T cells responding to peptide restimulation following LCMV reinfection: Fgl2 / and wild type mice were infected with 2x10 6 PFU of LCMV WE and at 120 days PI, these mice were reinfected with the same dose. Mononuclear cells were isolated from the spleen of wild type or fgl2 / mice on day 10 following reinfection. To assess the CD8 response, cells were re stimulated in culture for 6 hours in the presence of class I peptide GP33 or NP396 or with no peptide stimulation. Brefeldin A was added to the cell cultures to prevent cytokine secretion and the percentage of IFN γ+ of total CD8 + T cells was assessed by flow cytometry analysis. Values show the mean IFN γ+CD8 + cells / Total CD8 + cells ± SEM of 3 mice per group.
55 III.8 Conclusions:
These findings provide important information on the role for FGL2 in innate and adaptive immunity in a model of acute viral hepatitis caused by LCMV WE. Upon
LCMV infection of wild type mice, FGL2 prothrombinase activity is induced by macrophages, which was shown to limit viral dissemination within the liver. Secreted
FGL2 is also induced following viral infection and was shown to inhibit DC maturation.
By inhibiting DC maturation, both cellular and humoral responses were found to be decreased when compared with infected fgl2 / mice. Targeted deletion of FGL2 restores effective virus specific cellular and humoral responses in the primary challenge and secondary rechallenge.
56 CHAPTER IV
DISCUSSIO :
The LCMV WE model of acute viral hepatitis was utilized to evaluate the contribution of FGL2 in regulating the immune response towards viruses. An understanding of the regulatory role of FGL2 in both the innate and adaptive immune response will be critical to the development of novel therapeutic approaches to treat patients with viral hepatitis. These results collectively suggest that LCMV utilizes the
FGL2 FCγRIIB pathway to evade immune detection and that disruption of this pathway leads to enhanced maturation of DC, increased anti viral T cell responses in the periphery and the liver as well as increased production of both total and neutralizing antibodies to the virus.
LCMV has been studied extensively to gain insights into virus host interactions as a model of acute and chronic human hepatitis C virus infection 189 193 . LCMV is a single stranded RNA virus that belongs to the Arenaviridae family of viruses. Infection of mice with LCMV can result in an acute, protracted or lifelong infection depending on the strain of virus used and the genetic background of the host 194 195 . Viral clearance is dependent on robust cellular and humoral immune responses. The LCMV WE strain which was used in this study, is a viscerotropic virus causing acute liver disease in mice189 . Although most mouse strains clear LCMV WE within 2 weeks, mice demonstrate impaired innate and adaptive responses 196 197 .
We and others have recently reported that FGL2 is a putative effector molecule of
Treg, which plays an important role in the regulation of the immune response 112,149,153,212
214 . Antibody to FGL2 completely blocks the immunosuppressive activity of Treg and
57 restores proliferative T cell responses in a suppression assay 149 . Consistent with this, targeted deletion of fgl2 leads to impaired Treg function associated with enhanced reactivity of DC, T and B cells and manifestation of autoimmune kidney disease in aged fgl2 / mice 153 . Treatment with recombinant FGL2 inhibits the maturation of DC and effector T cell responses as well as induces B cell apoptosis 153 . FGL2 mediates its regulatory effects through binding to the inhibitory FcγRIIB receptor which is expressed primarily on APC, including DC and B cells 154 . In the absence of FcγRIIB, the suppressive effects of FGL2 on immune responses are abrogated 154 . Based on these studies, it was postulated that interference with the FGL2 FcγRIIB inhibitory pathway will result in enhanced anti viral immune responses in the experimental model of acute viral hepatitis caused by LCMV WE. To accomplish this, wild type and fgl2 / mice were utilized to examine the contribution of FGL2 to innate and adaptive immunity towards
LCMV 150 .
Macrophages are known to interact with LCMV early after infection and are critical role in regulating viral capture and elimination. In a study by Lang et. al, they showed that defects in macrophage function are associated with widespread dissemination of LCMV and subsequent T cell mediated immunopathology 197 . Consistent with the role of macrophages in controlling early viral spread, FGL2 expressed as a membrane associated prothrombinase on macrophages has also been previously shown to regulate viral spread in a murine model of MHV3 induced fulminant viral hepatitis. In
MHV3, fgl2 / mice had increased viral titres early in infection, despite having increased survival when compared to susceptible mice.
58 Therefore, to evaluate the role of membrane bound FGL2 in innate immune control of LCMV, FGL2 dependent thrombin generation in macrophages and viral titres were measured early after infection in wild type and fgl2 / mice. Following infection with LCMV, there was a dose dependent induction of FGL2 prothrombinase activity in wild type macrophages that is absent in fgl2 / macrophages ( Figure III 1.1 ). Consistent with the results found in the MHV3 model, increased liver viral titres were observed in fgl2 / mice during the early phase of LCMV infection ( Figure III 1.2 ). The loss of FGL2 expressed on the surface of reticulonendothelial cells may explain the enhanced viral replication and spread in fgl2 / mice compared to wild type mice during the early stage of
LCMV WE infection. These results also suggest that the prothrombinase activity of
FGL2 may contribute to the protective effects of macrophages early in infection.
Following infection with 2x10 6 PFU LCMV, enhanced maturation of DC in fgl2 / mice was observed as early as day 1 PI, as indicated by increased expression of the maturation markers CD80 and MHC II on CD11c + DC ( Figure III 4.1) . Previous studies from our laboratory have shown that DC from fgl2 / mice had enhanced maturation with increased expression levels of CD80 and MHCII in response to LPS stimulation 153 .
Fgl2 / mice also have increased numbers of splenic DC and activated DC from fgl2 / mice have increased survival 153
As early as 2 days PI, the levels of secreted FGL2 in the plasma of wild type mice were elevated, suggesting that FGL2 could act to regulate DC maturation at this timepoint (Figure III 2.1) . Moreover, the levels of secreted FGL2 peak 10 fold above naïve levels at day 8 PI and remain elevated from naïve levels until day 40 PI. This could indicate that FGL2 may exert its effects long after viral clearance in wild type mice and
59 may explain how FGL2 regulates the humoral response. Treg represent the subset of T cells that secrete the greatest amount of FGL2. Acute LCMV infection however, did not result in a significant expansion of Treg in both spleen and lymph node populations (data not shown) and the frequencies of Treg in the liver were constant until viral clearance at day 12 PI (data not shown). A number of studies suggest however, that the activity of
Treg producing anti inflammatory mediators is increased upon acute LCMV infection.
There are enhanced expression levels of the Treg effector molecules, IL10 215 216 and
TGF β217 in acute LCMV infection, albeit to lesser extents when compared to the levels observed in chronic LCMV infection. Thus, the increased activity of Treg can account for the increase in secreted FGL2 over the course of acute LCMV infection. Other possible sources include CD4 + and CD8 + T cells, as these have also been shown to secrete FGL2 in response to IFN γ stimulation. The dramatic increase in the number of splenic T cells in the context of an inflammatory response could account for the enhanced cumulative expression of FGL2 within the plasma, which coincidentally both peak at day 8 PI
(Figure III 2.1 ).
We propose that the increased maturation of DC in fgl2 / in the early stage of the infection contributes to the augmented anti viral T cell responses compared to wild type mice. In agreement with this hypothesis, there were increased frequencies of intrahepatic
CD8 + T cells and virus specific CD3ε +CD8α + GP33 + and CD3ε +CD8α + NP396 + T cells in mice deficient of FGL2 ( Figure III 5.1 ). In addition to the increased frequencies of virus specific T cells, the absence of FGL2 enhanced the responsiveness of both CD8 +
CTL ( Figure III 5.2 ) and CD4 + T helper cells ( Figure III 5.3 ) producing IFN γ in response to ex vivo peptide restimulation. Furthermore, recognizing the importance of
60 CD4 + T helper cells in providing appropriate cytokine stimulation, the increased function of helper T cell activity may further promote CTL responses and humoral responses.
These studies suggest that FGL2 in wild type mice inhibits the maturation of DC, which inhibits the function of virus specific cells responding to antigen and the trafficking of virus specific T cells to the liver.
In addition to its role in regulating T cell function by inhibiting DC maturation,
FGL2 was also found to regulate humoral responses in LCMV infection ( Figure III 6.1 ).
In previous in vitro studies, it was reported that binding of FGL2 to the inhibitory
FcγRIIB receptor leads to B cell and plasma cell apoptosis 154 . Consistent with these findings, it was shown that there were decreased numbers of CD138 +CD19 Low CD45R Low plasma cells following LCMV infection in wild type mice when compared to fgl2 / mice.
Consequently, the titre of both total LCMV specific and LCMV neutralizing antibodies also decreased in wild type mice in comparison to fgl2 / mice.
The increased expression of FGL2 also decreased antiviral activity within the liver when compared to fgl2 / mice in response to LCMV infection. Fgl2 / mice had increased inflammation within the liver as revealed by histological analysis and by increased levels of plasma ALT, a biomarker of liver necrosis ( Figure III 3.1). As with chronic HCV infection, the severity of liver inflammation correlates with the magnitude of protective CTL responses. Therefore, targeted deletion of fgl2 following LCMV WE infection leads to an enhanced intrahepatic immune response. Coincident with the induction of an enhanced inflammatory response, there was a reduction in viral titres to wild type levels, despite the 100 fold increase in viral titres that is observed on day 4 PI in fgl2 / mice ( Figure III 3.2 ). Moreover, high doses of LCMV are known to result in a
61 marked reduction in both CTL 218 221 and antibody responses 222 223 . Thus, despite the effects of immune paralysis, targeted deletion of FGL2 results in the generation of a robust CTL and humoral response towards LCMV.
The increased levels of FGL2 that were detected in the plasma of wild type mice following LCMV infection together with the enhanced immunity in fgl2 / mice suggest that FGL2 accounts for inhibition of DC maturation and consequently the impaired function of effector T and B cell anti viral responses. These findings are in agreement with the data reported on the immunoregulatory role of FGL2 in fulminant hepatitis caused by MHV3 149 . In the MHV3 model, we showed that increased plasma levels of
FGL2 and Treg cells correlated with susceptibility to the virus. Treatment with antibody to FGL2 blocked Treg cell activity and protected susceptible mice from liver injury and mortality induced by MHV3. Furthermore, adoptive transfer of wild type Treg cells into fgl2 / resistant mice increased their mortality following MHV3 infection 149 .
The effect of loss of FGL2 on generation of secondary immune response to
LCMV WE was examined in wild type and fgl2 / mice that were re infected with LCMV
120 days post primary infection. Fgl2 / mice were fully protected and did not develop biochemical or histological evidence of hepatitis at any time post LCMV WE re infection. In contrast, wild type mice developed a mild hepatitis which was confirmed by elevations in serum ALT, a marker of liver injury, as well as pathological signs of hepatic necrosis and inflammation (Figure III 7.1) . This difference could be explained by the fact that fgl2 / produced more vigorous primary and secondary anti viral immune responses to re infection with LCMV WE with significantly higher frequencies of virus
62 specific CD8 + T cells as well as higher titers of both neutralizing and non neutralizing anti LCMV antibodies compared to the wild type mice.
Recapitulating the results of the primary immune response, the secondary immune response to LCMV WE was also enhanced in fgl2 / mice at day 120 + 10 ( Figure III
7.2 ). However, the numbers of cells responding to ex vivo peptide restimulation at day
120 prior to reinfection were equivalent (data not shown). These results may suggest that the regulatory functions that promote contraction of the immune response, such as PD 1 and FAS L are still intact in fgl2 / mice. PD 1 / mice die due to overwhelming immunopathology when infected with LCMV Cl13 224 , due to a loss of immune regulation. Targeting FGL2 with antibodies directed against the immunosuppressive
FRED domain may safely enhance the efficacy of traditional antiviral therapy as it does not interfere with the termination of the immune response.
In this study we provided important insights into the biology of FGL2 in the context of acute viral hepatitis. We show that FGL2 plays important roles in enhancing innate immune reactivity, preventing widespread viral dissemination through the generation of thrombin and the deposition of fibrin at sites of infection. Moreover, the induction of secreted FGL2 by LCMV infection severely inhibits the induction of cellular and humoral immunity. Targeted deletion of fgl2 resulted in a restoration of immunity toward LCMV. Consequently, these studies provide fundamental insight into the efficacy of a therapeutic specifically targeting the FGL2 FCγRIIB pathway to treat immune dysfunction in chronic hepatitis viral infection and promote viral clearance.
LCMV Cl 13 contains mutations in the GP protein and polymerase gene, causing a persistent chronic hepatitis that has been used to model chronic HCV infection 184, 225 227 .
63 Consistent with the reports of others, we have shown that wild type mice infected with
LCMV Cl13 develop chronic hepatitis indicated by persistently elevated levels of serum
ALT ( Figure IV 1) with marked periportal and portal infiltration, lobular necroinflammation and bridging fibrosis detected by trichrome staining seen on day 28
PI. These changes to liver architecture persist for greater than 60 days ( Figure IV 1).
A
B *
C
Figure IV 1. Histopathological and biochemical features of LCMV Cl13 infection in wild type mice: C57BL/6 mice were infected intravenously with 2x10 6 PFU of LCMV Clone 13 and liver sections were stained (LEFT) using a hematoxylin and eosin (H&E) stain (A), Trichrome stains (B) on day 21 post infection (100x Magnification). Note presence of marked periportal lymphocytic infiltration (arrow) with piecemeal necrosis and bridging necrosis on the H&E staining (A). Trichrome staining of the same section revealed bridging fibrosis i.e portal porta area (arrow) (B). On day 28 post infection, H&E staining of the liver sections showed persistence of marked portal and periportal inflammation. Note formation of nodule surrounded by fibrosis (arrow) within the lobule (C). Serum ALT levels were also measured on days 15,21 and 28 post infection (RIGHT). Serum ALT levels increases by 5 fold at day 15 post infection compared to naive (uninfected) mice and remains elevated on days 21 and 28 post infection. Graph shows the mean± SEM of 3 mice per group; * P ≤ 0.05.
64 By plaque assay and immunohistochemistry, the virus is localized primarily in the cytoplasm and on the membrane of infected hepatocytes although virus was also seen in reticuloendothelial cells ( Figure IV 2).
In LCMV Cl 13 infection, effective T cell responses are crucial for clearance of viral infection and work to date has shown that in mice that develop chronic viral disease,
CTL responses are inhibited 71, 183, 228 233 . A recent report has demonstrated that Treg contribute to impairing CD8 + T cell responses to LCMV through modulation of DC activity 234 . The immunosuppressive molecules PD 1, CTLA 4 and IL 10 have been suggested to account for the sustained suppression of CD8 + T cells during persistent
LCMV infection 205, 235 236 , although the exact mechanisms that initially induce immunosuppression and lead to the loss of T cell cytolytic and stimulatory function are presently unknown. In addition, blockade of these inhibitory regulators only partially restores anti viral immunity suggesting that other inhibitory pathways/molecules are involved in viral persistence and loss of immunity 198, 235 236 .
To evaluate the contribution of FGL2 to the immunosuppression in chronic hepatitis infection, we have initiated preliminary studies using LCMV Cl13. In agreement with the enhanced virus induced immunosuppression that is observed in persistent
LCMV CL13 infection, plasma FGL2 levels in mice infected with LCMV Cl13 were found to be significantly elevated when compared to the levels observed during LCMV
WE infection (Figure IV 3), peaking at day 10 PI with a concentration of 44.8 ± 2.3 ng/mL. Moreover, the levels of FGL2 gradually decreased through the course of infection, but never returned to naïve levels at all time points measured. A recent report from Wherry and Ahmed has shown that mice chronically infected with LCMV Cl13
65 have increased expression of mRNA for fgl2 and FCγRIIB in functionally impaired virus specific CD8 + supporting our hypothesis that the FGL2 FCγRIIB inhibitory pathway plays a critical role in inhibiting antiviral T cell responses 236 .
Liver Viral Titres In Wild Type Mice infected with 2x10^6 PFU of LCMV Clone 13
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01 Viral Titres (PFU/g Liver) (PFU/g Viral Titres 1.00E+00 Day 15 Day 21 Day 28
.
Figure IV 2. Viral titres in wild type mice following LCMV Cl 13 infection: Representative picture of LCMV clone 13 nucleoprotein immunostaining on day 21 post infection in the liver of infected mice ( LEFT ). C57BL/6 mice were infected intravenously with 2x10 6 PFU of LCMV Cl 13. Mice were sacrificed on day 21. Liver sections were cut, frozen and stained for the LCMV nucleoprotein using VL4 ascites fluid at a dilution of 1:50. Note that LCMV nucleoprotein was detected essentially in the hepatocytes (arrow) but also within the non parenchymal cells. Mice were sacrificed at various time points and liver titers were determined using an LCMV focus forming assay (RIGHT ). Viral titers were elevated in the liver of infected mice at various time points studied; Graph shows the mean± SEM of 5 mice per group.
66
50 45 40 35 30 25 20 15 FGL2 Concentration FGL2 10
5
0
Naïve Day 5 Day 10 Day 15 Day 20 Day 28
Figure IV 3. FGL2 is induced in vivo upon LCMV Cl13 infection and remains persistently elevated: C57BL/6 mice were bled at various time points following infection with 2x10 6 PFU of LCMV Cl 13. FGL2 was measured in plasma by ELISA. Values shown are the mean FGL2 concentrations of 5 mice per time point and are expressed as mean ± SEM.
In addition to the studies in mice, we have also initiated studies in patients with chronic HCV infection. The concentration of FGL2 was found to be elevated in patients with chronic HCV infection compared to healthy controls or to patients with alcoholic cirrhosis and HCV patients who received antiviral therapy and attained SVR ( Figure IV
4). We have further demonstrated that serial measurements of FGL2 levels during the course of antiviral therapy showed a greater decrease from baseline measurement in
67 patients who achieved a SVR and control viral infection when compared to non responders and relapsers. In a small number of patients we have also shown prospectively that increased plasma levels of FGL2 in HCV patients is associated with unresponsiveness to therapy 237 .The elevated levels of FGL2 observed in chronic LCMV
Cl 13 and HCV infection suggest that FGL2 may contribute to viral persistence in chronic infection. Further studies are now underway in a chronic model of LCMV Cl13 infection and human viral hepatitis. The preliminary studies all suggest that secreted
FGL2 may be responsible for inhibiting the immune response in chronic hepatitis infection and that targeting secreted FGL2 may then ultimately help to restore immunity and promote viral clearance.
Figure IV 4. Mean plasma levels of FGL2 in patients with chronic HCV infection: 10 ml of heparinized blood was collected from 80 patients with chronic HCV infection, which had not received anti viral therapy. Mean plasma levels of FGL2 in these patients were compared to 30 healthy controls, 24 patients with inactive alcoholic cirrhosis, and 32 patients with chronic HCV who cleared the virus following successful anti viral therapy (SVR). Mean plasma levels of FGL2 were significantly higher in patients with chronic HCV infection.
68 IV.1 Future Directions:
The results of these preliminary studies have shown that FGL2 is highly induced in chronic infections, such as HCV and LCMV Cl13, when compared to acute infection.
It is hypothesized that FGL2 mediated immune suppression contributes to persistence in chronic LCMV and HCV infection. To evaluate the role of FGL2 in establishing chronic infection, we propose to infect fgl2 / mice and fcγrIIb / mice with LCMV Cl13.
Following infection, viral titres will be measured for clearance, as well as the cellular and humoral immune response. Following infection, we hypothesize that disruption of the
FGL2 FCγRIIB pathway results in enhanced cellular and humoral immune responses toward LCMV, which will result in viral clearance of LCMV Cl13 after induction of the adaptive immune response. In addition to experimentation in knockout models, these studies should be reproduced in wild type mice treated with antibody towards FGL2 and infected with LCMV Cl13. These experiments would be critical in the evaluating the effect of blocking the FGL2 FCγRIIB pathway in chronic infection in the absence of the various compensatory mechanisms that can confound results in various knockout models.
A key goal in these studies is to evaluate the contribution of the FGL2 FCγRIIB pathway in the persistence of human HBV and HCV infection. Polymorphisms in the
FcγR and fgl2 have been identified and these may contribute to or confer resistance to chronic HCV infection. Correlating such polymorphisms to clinical outcome, disease severity and virologic response in patients infected with HCV may also be predictive of response to antiviral therapy. If the proposed studies are successful in humans, the measurement of FGL2 plasma levels and evaluating the polymorphisms in FcγR and fgl2 genes will become a standard of care in the medical management of chronically infected
69 HBV and HCV patients. Furthermore, blocking the FGL2 FCγRIIB pathway with antibodies may provide a novel approach to improve virus eradication and long term patient outcomes in chronic HCV infection.
70 CHAPTER V
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