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Transmitted/founder hepatitis C viruses induce cell-type- and genotype-specific differences in innate signaling within the liver

Significance Statement

Hepatitis C virus (HCV) infection is the most common chronic viral infection for which there is no vaccine available; the immune response to Hepatitis C virus remains incompletely elucidated.  There are multiple genotypes of the virus, and it is known that patients respond to antiviral therapy differently depending on which genotype of the virus they are infected with. HCV reaches the liver via the portal vein or the hepatic artery and infects hepatocytes, the known major site of replication. Hepatocytes comprise about two-thirds of the total cell population within the liver; the remaining population of non-parenchymal liver cells (NPCs) is diverse, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), hepatic stellate cells (HSCs), and intrahepatic lymphocytes.  Despite not sustaining productive viral replication, these NPCs are likely to sense Hepatitis C virus RNA and shape the outcome of infection.  We reasoned that the cellular site of initial infection might play a critical role in regulation of HCV replication within hepatocytes. We tested full-length HCV transmitted/founder (T/F) genomes possessing sequences that favor efficient in vivo replication and are responsible for transmission and productive infection. We found that hepatitis C T/F viruses induced robust transcriptional and protein expression of interferons (IFNs), Type I and III.  Interestingly, liver endothelial cells which compose the highest proportion (approximately 50%) of the non-parenchymal cells, produced even higher levels of IFN.  The monocytic cell line THP-1 and primary macrophages were noted to produce higher IFN levels with genotype 3a than genotype 1.  We subjected primary human hepatoycte lysates after 8 hours of transfection with either genotype 1a or 3a T/F vRNA to transcriptomic microarray analysis. We found that genotype 3a induced significantly higher gene expression of CXCL10, CXCL11, EGR1 (early growth response protein 1), and IFIT3 (interferon-induced protein with tetratricopeptide repeats 3).

What are the clinical implications of these findings?

We found that the immune response varied according to both cell type and Hepatitis C virus genotype, leading to a more pronounced induction of inflammatory pathways after exposure to certain genotypes. Our study supports the idea that inflammatory pathways that are being robustly activated by certain Hepatitis C virus genotypes could lead to more severe damage (including development of fibrosis and hepatocellular carcinoma)  and demonstrate variable response to antiviral therapies.

Transmitted/founder hepatitis C viruses induce cell-type- and genotype-specific differences in innate signaling within the liver.. Global Medical Discovery

 

 

 

 

 

 

Journal Reference

Mitchell AM, Stone AE, Cheng L1, Ballinger K2, Edwards MG3, Stoddard M4, Li H4, Golden-Mason L, Shaw GM4, Khetani S2, Rosen HR5. MBio. 2015 Feb 24;6(2):e02510.

Show Affiliations

1Division of Gastroenterology and Hepatology, Hepatitis C Center, Department of Medicine, University of Colorado Denver, Denver, Colorado, USA.

2Mechanical and Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA.

3Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, Colorado, USA.and

4Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

5[email protected]

 Abstract

Hepatitis C virus (HCV) infection leads to persistence in the majority of cases despite triggering complex innate immune responses within the liver. Although hepatocytes are the preferred site for Hepatitis C virus replication, nonparenchymal cells (NPCs) can also contribute to antiviral immunity. Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference have identified full-length transmitted/ founder  (T/F) viruses. Here, we tested the effect of HCV T/F viral RNA (vRNA) on  innate  immune  signaling within hepatocytes and NPCs, including the HepG2 and Huh 7.5.1 cell lines, a human liver endothelial cell line (TMNK-1), a plasmacytoid dendritic cell line (GEN2.2), and a monocytic cell line (THP-1). Transfection with hepatitis C T/F vRNA induced robust transcriptional upregulation of type I and III interferons (IFNs) within HepG2 and TMNK-1 cells. Both the THP-1 and GEN2.2 lines demonstrated higher type I and III IFN transcription with genotype 3a compared to genotype 1a or 1b. Supernatants from HCV T/F vRNA-transfected TMNK-1 cells demonstrated superior viral control. Primary human hepatocytes (PHH) transfected with genotype 3a induced canonical pathways that included chemokine and IFN genes, as well as overrepresentation of RIG-I (DDX58), STAT1, and a Toll-like receptor 3 (TLR3) network. Full-length molecular clones of HCV induce broad IFN responses within hepatocytes and NPCs, highlighting that signals imparted by the various cell types within the liver may lead to divergent outcomes of infection. In particular, the finding that Hepatitis C virus genotypes differentially induce antiviral responses in NPCs and PHH might account for relevant clinical-epidemiological observations (higher clearance but greater necroinflammation in persistence with genotype 3).

IMPORTANCE:

Hepatitis C virus (HCV) has become a major worldwide problem, and it is now the most common viral infection for which there is no vaccine. HCV infection often leads to persistence of the virus and is a leading cause of chronic hepatitis, liver cancer, and cirrhosis. There are multiple genotypes of the virus, and patients infected with different viral genotypes respond to traditional therapy differently. However, the immune response to the virus within the liver has not been fully elucidated. Here, we determined the responses to different genotypes of HCV in cell types of the liver. We found that the immune response varied according to both cell type and HCV genotype, leading to a more pronounced induction of inflammatory pathways after exposure to certain genotypes. Therefore, inflammatory pathways that are being robustly activated by certain HCV genotypes could lead to more severe damage to the liver, inducing diverse outcomes and responses to therapy.

Copyright © 2015 Mitchell et al.

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