Development and use of in vitro systems for the investigation of the immunomodulatory properties pf core/core+1 proteins of the hepatitis C virus (HCV)

Hepatitis C virus is a major public health issue, as 170 million people are infected worldwide, and 2 million new cases are reported each year. 80% of the infected individuals develop persistent infection, which results in liver cirrhosis and hepatocellular carcinoma. The goal of this thesis is the development and use of in vitro systems for the investigation of the immunomodulatory properties of the virus and in particular of the capsid protein Core and the newly discovered Core+1. Our results show that both Core and Core+1 interfere with the production of certain cytokines (IL-6, IL-10, TNF-α) and chemokines (MCP-1α, MIP-1B, IL-8) by the liver cells, in addition to cells of the immune system, such as macrophages. This interaction could be performed through interference with promoters, important for the innate immune response and elements such as NF-kB element. Conclusively, our results provide evidence for the participation of Core and Core+1 proteins in the immunomodulatory properties of the virus.Ο ιός της ηπατίτιδας C αποτελεί μείζον πρόβλημα της δημόσιας υγείας καθώς εκτιμάται ότι 170 εκατομμύρια άνθρωποι είναι μολυσμένοι, ενώ 2 εκατομμύρια νέα κρούσματα προστίθενται κάθε έτος. Στο 80% των περιπτώσεων ο ιός εγκαθιστά χρόνια μόλυνση, η οποία μπορεί να καταλήξει σε κίρρωση του ήπατος και ηπατοκυτταρικό καρκίνο. Στόχος της παρούσας διατριβής είναι η ανάπτυξη και χρήση in vitro συστημάτων για την διερεύνηση των ανοσορυθμιστικών ιδιοτήτων του ιού και πιο συγκεκριμένα της καψιδιακής πρωτεΐνης Core και της καινούργιας πρωτεΐνης Core+1. Παρατηρήθηκε ότι τόσο η Core πρωτεΐνη όσο και η Core+1 εμπλέκονται στην παραγωγή κυτοκινών (IL-6, IL-10, TNF-α) και χημειοκινών (MCP-1α, MIP-1B, IL-8) από τα ηπατικά κύτταρα, αλλά και από κύτταρα του ανοσοποιητικού συστήματος, όπως τα μακροφάγα. Η επίδραση αυτή μπορεί να πραγματοποιηθεί και μέσω της επιρροής της ενεργότητας υποκινητών σημαντικών στη φυσική ανοσία και αλληλουχιών πρόσδεσης μεταγραφικών παραγόντων όπως του NF-kB. Μάλιστα, η επίδραση αυτή φαίνεται να εξαρτάται από τον γονότυπο του ιού με τον οποίο είναι μολυσμένος ο ασθενής. Συμπερασματικά τα αποτελέσματα αυτά παρέχουν ενδείξεις για την ανοσορυθμιστική ικανότητα τόσο της Core όσο και της Core+1 πρωτεΐνης και ενισχύουν την άποψη για συμμετοχή των πρωτεϊνών αυτών στην προσπάθεια του ιού να εγκαταστήσει μόνιμη μόλυνση και χρόνια φλεγμονή, αποφεύγοντας τις ανοσολογικές αποκρίσεις του ξενιστή

Modulation of monocyte/macrophage-derived cytokine and chemokine profile by persistent Hepatitis C virus (HCV) infection leads to chronic inflammation

HCV infection presents a major public health problem, with more than 170 million people infected worldwide. Chronicity and persistence of infection constitute the hallmark of the disease. Although HCV is a hepatotropic virus, subsets of immune cells have been found to be permissive to infection and viral replication. Peripheral blood monocytes, attracted to the site of infection and differentiated into macrophages, and resident hepatic macrophages, known as Kupffer cells, are important mediators of innate immunity, through production of several chemokines and cytokines in addition to their phagocytic activity. HCV proteins have been shown to modulate the cytokine and chemokine production profile of monocytes/macrophages, as it is suggested by both in vitro and clinical studies. This modified expression profile appears crucial for the establishment of aberrant inflammation that leads to liver cirrhosis and hepatocellular carcinoma

HCV-Induced Immunometabolic Crosstalk in a Triple-Cell Co-Culture Model Capable of Simulating Systemic Iron Homeostasis

Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1 alpha activity

Background & Aims: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR-and HNF-1 alpha-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. Methods: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. Results: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1 alpha element in ANGPTL-3 promoter was mutated, while loss of HNF-1 alpha DNA binding to this site was recorded in the presence of HCV core. HNF-1 alpha mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1 alpha-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore coremediated down-regulation of ANGPTL-3 promoter activity. Conclusions: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1 alpha binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved