Principal Investigator: Prof. Adriana Vince, MD, PhD
Host institution: University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”, Mirogojska 8, 10000 Zagreb, Croatia, web: www.bfm.hr , mail: bfm@bfm.hr , tel 385 1 2826 222

PROJECT TEAM MEMBERS
(Doctor”s degree) Snježana Židovec Lepej, (Bachelor”s degree) Lana Gorenec, (Doctor”s degree) Ivana Grgić, (Bachelor”s degree) Vjeran Čajić, (Doctor”s degree) Ivan Kurelac, (Bachelor”s degree) Neven Papić, (Bachelor”s degree) Davorka Dušek, (Doctor”s degree) Tajana Filipec Kanižaj

BUDGET: 1,000,000.00 kn
FUNDING: HRZZ – Croatian Science Foundation
PROJECT DURATION: Starting date: 01.07.2014. Finishing date: 30.06.2018. (48 months)

PROJECT SUMMARY
Hepatitis C virus (HCV) is the leading cause of liver disease. HCV productively infects hepatocytes to impart liver inflammation and progressive tissue damage leading to fibrosis and cirrhosis. These processes underlie liver dysfunction and are thought to drive the onset of liver cancer. However, the molecular mechanism(s) by which HCV confers hepatic inflammation are not defined. Now, there is growing evidence that liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC), may play key roles in regulating immune responses and facilitating tolerance induction. These cells are playing a pivotal role in blood-borne virus clearance (>90%), leaving only a small fraction of infectious virus that escapes clearance in a manner peculiar to each individual pathogen. The biology of HCV, specifically regarding non-parenchymal liver cells, has been largely neglected. LSEC account for the 20% and KC for 15% of the hepatic cells, and are a unique organ-resident cell population with diverse functions, including degradation of bacterial by-products, antigen presentation and induction of tolerance. The hypothesis of the research is that HCV infection reprograms cellular gene expression of LSEC and KC, which results in the modulation of the inflammatory responses. Applying the infectomics approach, we plan to perform a series of next-generation sequencing experiments on LSEC and KC isolated and purified from healthy, mild and severe chronic hepatitis C liver biopsies. This will allow us to detect expression changes related with disease progression. Selected candidate genes will be comprehensively studied in in vitro systems in order to elucidate their role in modulation of inflammatory and adaptive immune responses. Through the development of this project it is expected that a series of high impact results will be obtained, which will facilitate the development of more significant biomarkers and effective interventions against HCV infection.

HYPOTHESIS and OBJECTIVES
The hypothesis of the research is that HCV infection reprograms cellular gene expression of human liver sinusoidal endothelial cells and Kupffer cells which results in the modulation of the inflammatory responses. The goal is to identify new modulators of inflammatory responses by using the newly developed infectomics approach.
The overall goal of the project is to detect, by sequencing the LSEC and KC transcriptomes of HCV infected individuals, pathogenic alterations in the gene expression of these patients through a comparison with healthy individuals, using the next generation RNA-seq technology. This will allow us to identify new regulators of inflammation, which will facilitate the development of more significant biomarkers, effective interventions against HCV infection and might aid the development of novel strategies for the design of vaccines against HCV infection.
General Aim: To determine the transcriptome associated with immune response in LSECs and KC, selected from liver biopsy samples from different stages of chronic hepatitis C disease, and to apply the dataset toward understanding the molecular processes of the inflammatory response against HCV.
Specific Aims: (1.)to isolate and characterize human LSEC and KC from livers of patients with chronic hepatitis C infection; (2.) to identify changes in LSEC and KC gene expression patterns in different stages of HCV liver disease using next-generation RNA-seq technology; (3.) to propose the molecular mechanism of new regulators of inflammation. This includes: a) to select and study candidate genes, previously unrelated to HCV infection; b) to determine the role of selected genes on inflammatory cytokines production; c) to investigate whether LSEC/KC regulates T-cell responses; (4) to identify, validate and explore potential new biomarkers and their clinical adaptability.

EXPECTED RESULTS
Using system biology approach we will identify new KCs and/or LSECs genes and host-specific pathways related with HCV pathology. The increasing understanding of the molecular interactions between the virus and its host cell will allow the identification of promising host targets for anti-HCV therapy. We will characterize changes in LSECs and KCs in different stages of liver disease, and provide new evidence on their role in HCV (and possibly other viral infections) pathogenesis. This will lead us to identification of new proteins that can easily be measured in the patients’ serum. Several markers will be validated across different fibrosis stages and compared to the markers used in currently available tests (e.g. FibroTest, ELF test, FIBROSpec), which have difficulties in classifying the intermediate stages of fibrosis often referred to as “gray area”, therefore leaving a demand for more reliable non-invasive markers to decrease the need for liver biopsy. Thus, there is a reasonable chance for further collaboration with pharmaceutical and clinical diagnostics industry.
The innovatory significance of this project lies in its focus on whole-genome sequencing-based HCV-host response profiling. Remarkably, the number of published papers using genomics approaches in the infectious diseases setting still lags significantly behind the number of papers on the use of this approach in oncology. The methodology of the project is innovative since it brings together systemic biology and conventional approaches directly to the traditional patient-based infectious diseases studies. There is an urgent need for holistic approaches that can integrate the dynamic interactions between microbial pathogens and their hosts during the development of infectious diseases. Here, the newly developed infectomics enable us to efficiently, and integratively study functional genomics of HCV infection.
To the best of our knowledge, this will be the first comprehensive gene expression and biomarker study on human KC (gene expression profile have not been published yet) and LSEC, in the natural course of HCV infection; therefore it has a very high significance for HCV research. Through the development of this project it is expected that a series of high impact results will be obtained: (1) significant increase in knowledge of the LSEC and KC roles in HCV and possibly other viral infections; (2) annotation of the structure and function of newly discovered human genes; (3) development of new bioinformatics tools (4) generation of new knowledge and leadership in biomedical research in fundamental aspects of cell and molecular biology related to infectious diseases; (5) development of new HCV disease models; (6) development of new biomarkers and treatment possibilities. This approach is likely to have general applications in studying viral-host interactions in both model systems and clinical specimens.