Innate immunity in hepatitis C infection and fibrosis


Global microRNA profiling reveals complex interactions among hepatic microRNA regulation, hepatitis C virus infection and interferon response

Hawwa F. Alao, Helen Cha, Stephan Chiu, Qisheng Li, T. Jake Liang

Liver diseases branch, NIH, Bethesda, MD

MicroRNAs (miRNAs) are small non-coding RNAs that fine tune gene expression to control essential biological processes through down-regulation of translation or transcription of mRNAs. Host miRNAs, like miR-122, have been shown to play an important role in hepatitis C virus (HCV) replication. HCV, on the other hand, may manipulate miRNA expression in infected hepatocytes to create a favorable host environment for productive infection and propagation. We recently conducted a genome-wide functional screen and identified an entire repertoire of cellular miRNAs that are associated with the complete life cycle of HCV. To further investigate the interactions between host miRNAs and HCV, we performed global miRNA expression analyses in both primary human hepatocytes and Huh. 7.5.1 human hepatoma cell line. Cells were infected with HCV at various time points or treated with interferon-alpha (IFNalpha) or interferon-lamda (IFN-lambda) either in the presence or absence of HCV infection. Applying the Nanostring miRNA profiling technology, we identified multiple miRNAs that were significantly regulated by HCV infection or interferon treatment. HCV treated cells showed an overall decrease in general microRNA expression at all time points, albeit several miRNAs were considerably up-regulated by HCV. These HCV-induced miRNAs include miR-122, miR-107, miR-29a-3p, miR-27b-3p and miR-301a-3p. Increased expression of miR-122 in HCVinfected cells aligns with a proviral role of the miRNA in HCV replication. Interestingly we showed that IFN-alpha generally decreased the overall miRNA expression levels, whereas IFNlambda increased the general microRNA expression, suggesting that distinct mechanisms may be engaged by these two families of IFNs to regulate miRNA profiles in hepatocytes. Among the IFN-modulated specific miRNAs are let 7b-5p, miR 425-5p, miR 140-5p, miR 1066-5p and miR 125b-5p. Conclusion: HCV infection induces a unique response in miRNA expression to facilitate productive infection. This response may result from a complex interplay among innate mechanisms, such as interferon responses, in infected hepatocytes. A comprehensive study of host miRNA expression and regulation associated with HCV infection may provide crucial insights into HCV-host interactions and mechanisms of interferon response.


The following people have nothing to disclose: Hawwa F. Alao, Helen Cha, Stephan Chiu, Qisheng Li, T. Jake Liang


HCV induces differentiation of CD14+ circulating monocytes via TLR8 into macrophage-like cells that exhibit M2 (alternative) polarization and profibrogenic effects

Banishree Saha, Gyongyi Szabo

Medicine, UMASS Medical School, Worcester, MA

Introduction: Macrophage activation and dysfunction contribute to chronic hepatitis C virus (HCV) infection and liver fibrosis. However, the nature of macrophage (MΦ)polarization during HCV infection is not known. Depending on the signals from the tissue microenvironment, circulating monocytes differentiate into MOs with either MI(classical) or M2 (alternative) polarization. We hypothesized that HCV provides signals for circulating monocytes to differentiate into “pathogenic macrophages〃+ Methods: Surface markers of circulating monocytes from HCVinfected patients and controls were studied by flow cytometry. In vitro co-culture experiments were performed with monocytes isolated from healthy donors (n=1 0-15) and hepatoma cells (Huh7.5) infected with HCV (JFH-1/ Huh7.5). Results: We found that circulating monocytes from chronic HCV-infected patients exhibit an M2 polarized phenotype with high expression of CD206 (mannose receptor) and CD163 (scavenger receptor) proteins as compared to healthy controls. Further, transcriptional analysis of liver biopsies from chronic HCV patients revealed an increase in M2 MΦ marker (CD206, IL-10 and TGF-β) expression as compared to control livers. We observed that HCV-infected hepatoma cells (JFH-1/Huh7.5) induced differentiation of normal monocytes to MΦ-like cells in vitro. These ˝HCV-educated˝MΦs displayed increased expression of M2 markers with no change in the M1 marker expression. Monocytes co-cultured with JFH-1/Huh7.5 cells secreted pro-inflammatory (IL-1 p and TNF-a) and predominantly antiinflammatory (IL-10 and TGF-β) cytokines. We further observed that early secretion of IL-1 p facilitated TGFβ secretion, as this process was inhibited by IL-1 receptor antagonist, anakinra. The high level of TGF-β secreted by ˝HCV-educated˝ MΦ was pro-fibrotic and led to activation of hepatic stellate (LX2) cells as this process could be blocked by anti-TGFβ neutralizing antibody. Transwell co-culture experiments revealed that monocyte differentiation was induced by cell-free or exosome-bound HCV and did not require contact with JFH-1/Huh7.5 cells. Finally, we discovered that TLR8 stimulation induced monocyte to M2 MΦ differentiation and that HCV triggered monocytes to differentiate into M2 MΦ-like cells via the TLR8 receptor as TLR8 knockdown prevented HCV-induced monocyte differentiation.

Conclusion: We describe a mechanism wherein HCV interacts with circulating monocytes and induces TLR8-mediated differentiation towards an anti-inflammatory, M2 MΦ-like phenotype that promotes liver fibrosis. This study provides novel insights into the mechanism by which HCV evades the host immune system and induces liver fibrosis.


The following people have nothing to disclose: Banishree Saha, Gyongyi Szabo


Monocytes Regulate NK Cell Antiviral Function in Hepatitis C via Inflammasome-Induced IL-18

Elisavet Serti1, Jens M. Werner1, Michael A. Chattergoon2, Andrea Cox2, Volker Lohmann3, Barbara Rehermann1

1Immunology Section, Liver Diseases Branch, NIDDK, National Institutes of Health, Bethesda, MD; 2Division of Infectious Diseases, Johns Hopkins School of Medicine, Baltimore, MD; 3Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany

BACKGROUND & AIMS: Natural killer (NK) cell IFN-γ production is impaired in chronic HCV infection. Here, we asked whether this impairment is NK cell-intrinsic or extrinsic. METHODS: Hepatoma cells expressing luciferase-tagged subgenomic HCV replicons (Huh7/HCV-replicons) or their HCV-negative counterparts (Huh7) were co-cultured with NK cells in the presence or absence of other PBMC subpopulations. Antiviral activity, cytotoxicity, and cytokine production were assessed. RESULTS: NK cells exerted greater IFN- γ responses (38% vs 22% IFN- γ + NK cells, p=0.0038; MFI 369 vs 186, p=0.0039) but minimal target cell killing (11% vs. 0.5%, p<0.05) when PBMC were co-cultured with Huh7/HCV-replicons compared to co-culture with Huh7. These results were consistent with higher IFN- γ (371 vs. 183 pg/ml, p=0.016) and TNF-α levels (947 vs. 486 pg/ml, p=0.016) in the culture supernatant. Neutralization of IFN- γ and TNF-α significantly increased HCV replication. However, IFN- γ production and antiviral function of NK cells were significantly lower when isolated NK cells rather than PBMC when co-cultured with Huh7/HCV-replicons (24% vs. 71% decrease in replication) suggesting that other PBMC subpopulations contributed to NK cell activation. Increased levels of multiple monokines suggested a role of monocytes in NK cell activation. This was supported by an increased expression of the activation markers HLA-DR (MFI 33398 vs. 28040, p=0.008) and CD69 (MFI 745 vs. 589, p=0.004) on CD14+CD16+ monocytes in co-cultures with Huh7/HCV-replicons compared to co-cultures with Huh7 cells. In addition, depletion of monocytes diminished the NK IFN- γ response (9% vs. 20.7% IFN- γ + NK cells, p< 0.0001; MFI 127 vs. 277, p=0.002). Furthermore, siRNA knockdown of the NALP3 inflammasome in primary human monocytes decreased the frequency and the MFI of IFN- γ producing NK cells (14% vs. 25. 8%, p=0.031; MFI 304 vs. 440, p=0.003), as did the neutralization of the inflammasome product IL-18 (22% vs 43%, p=0.03; MFI 280 vs 555, p=0.031). Finally, monocytes from chronic HCV patients were less effective than monocytes from healthy controls in stimulating the antiviral function of healthy blood donor NK cells (55% vs. 71% decrease in replication, p=0.011). Vice versa, monocytes from healthy controls improved the antiviral activity of NK cells from chronic HCV patients (75% vs. 61% decrease in replication, p=0.019). CONCLUSIONS: Monocytes sense HCV-replicating cells and trigger, via inflammasome activation and IL-18 production, both IFN- γ secretion and antiviral activity of NK cells. An impaired monocyte function contributes to the suboptimal IFN- γ production of NK cells in chronic HCV infection.


The following people have nothing to disclose: Elisavet Serti, Jens M. Werner, Michael A. Chattergoon, Andrea Cox, Volker Lohmann, Barbara Rehermann


Tissue macrophages are responsible for inflammatory liver disease in the hepatitis C virus transgenic mice

Kiminori Kimura1,2, Takahiro Ohtsuki2, Yuko Tokunaga2, Michinori Kohara2

1Division of Hepatology Tokyo Metropolitan Cancer and Infectious diseases Center Komagome Hospital, Byunkyo-ku, Japan; 2Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Japan

Purpose: Tissue macrophages are widely defined as important inflammatory cells to chronic viral hepatitis due to their proinflammatory activity. We have already reported that hepatitis C virus transgenic mice (HCV Tg) caused continuous liver injury and developed hepatocellular carcinoma through the Cre/loxP switching system (Blood, 2010). In addition, we showed recombinant vaccinia viruses expressing HCV nonstructural protein (rVV-N25) could protect against the progression of chronic hepatitis by way of suppression of macrophages activation. Herein, we focused on the role of tissue macrophages for liver disease of the HCV Tg mice and examined characteristic features of macrophages following rVV-N25 treatment. Methods: First, to determine whether HCV protein expression affects on tissue macrophages accumulation we isolated mononuclear cells in the liver, spleen and stromal vascular fraction (SVF) of adipose tissue from HCV Tg mice. The mononuclear cells were analyzed by FACS using macrophages marker (CD11b, CD11c, CD206, Gr-1, F4/80, Ly6C) and the cytokines production (TNFa, IL-6, arginase). Further, we compared with tissue macrophages number and cytokine production in the same tissue following rVVN25 treatment. To confirm the effect of tissue macrophages in liver disease of HCV Tg mice, we were injected intravenously with clodronate liposomes and examined serum ALT activity, HCV core protein level, cell number and observed histological features. Finally, we analyzed liver disease and the function and number in tissue macrophages with Tg mice following neutralizing anti-IL-6R antibody. Results: The number of CD11b+F4/80+macrophages in the liver and spleen but not SVF was increased under HCV expressed condition and especially CD11b+F4/80+CD11c-CD206+M2 macrophages remarkably increased. In addition, these M2 as well as M1 macrophages were produced TNF-a and IL-6 much higher with HCV Tg mice. rVV-N25 treatment suppressed cell number and cytokine production on macrophages in the liver and spleen. However, TNF-a production from M2 macrophages was increased in the SVF. We also showed that pathological findings in the liver have improved by depletion of macrophage even though HCV core level was not suppressed. Finally, aIL-6R antibody treatment reduced the number of macrophages and induced normal pathological findings. Conclusions: M2 macrophages contribute to the induction of chronic liver inflammation in HCV mouse models. In addition, rVV-N25 treatment induced therapeutic effect on liver tissue due to suppressed macrophage recruitment and activation.


The following people have nothing to disclose: Kiminori Kimura, Takahiro Ohtsuki, Yuko Tokunaga, Michinori Kohara


IL-4 Receptor alpha1 regulates M2 macrophage signaling and is an important modulator of liver fibrosis progression and regression

Shih-Yen Weng1, Kornelius Padberg1, Yong Ook Kim1, Xiao-Yu Wang1, Brombacher Frank2, Detlef Schuppan1,3

1Institute of Molecular and Translational Medicine, University, Medicine, Johannes Gutenberg University Mainz, Germany; 2International Center for Genetic Engineering and Biotechnology, Cape Town, South Africa; 3Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Liver fibrosis progression and regression are modulated by cells of the innate immune system, especially macrophages. Macrophages can be roughly classified as classically activated and alternatively activated (M1 and M2, respectively). While M2 have been implicated in fibrogenesis, the overall functional role of M1 and M2 in liver fibrosis remains largely unknown. M2 polarization is controlled by signaling transmitted through IL-4 and IL-13 ligation to receptors IL-4Ra1 (and IL-13Ra1). This correlates with upregulation of M2-related genes such as Stat6, Arg1, YM1 and Mrc1. We therefore aimed to define the role of IL-4Ra1 as central receptor for M2 polarization in liver fibrosis progression and spontaneous recovery. We used the mouse models of CCl4-induced (6 weeks by oral gavage) and spontaneous biliary liver fibrosis (Mdr2 KO). In the CCL4 model, IL4Ra1 expression was increased >2-fold after 6 weeks and reduced 1.5-fold 2 weeks after the last dose. In Mdr2 KO mice, IL-4Ra1 expression gradually increased until age 6-wk, and decreased thereafter. To address the function of IL-4Ra1 in vivo, Balb/c mice with systemic and macrophage-specific deletion of IL-4Ra1 (IL-4Ra1 ΔLysM) were used. IL-4Ra1 KO mice showed a significantly decreased expression of M2 markers such as YM1 throughout all stages of fibrosis progression and reversal. Using Sirius red staining and biochemical quantification, CCL4treated mice with systemic and cell specific IL4Ra1 knockout showed significantly less collagen accumulation compared to wildtype controls during fibrosis progression. This was rversed during fibrosis regression. Compared to wildtype littermate controls, IL-4Ra1 ΔLysM but not systemic IL-4Ra1 KO mice showed an attenuated ALT elevation. Interestingly, macrophage markers like CD68, IL-1b, Arg1 and fibrosis related genes such as procollagen α1(I) and αSMA were significantly downregulated in IL-4Ra1 ΔLysM and systemic IL-4Ra1 KO mice during fibrogenesis, but upregulated during fibrosis regression. We show a central role of IL-4Ra1 signaling in M2 macrophage polarization, with a profibrotic role during liver fibrosis progression and a fibrolytic role during its regression. Modulation of IL-4Ra1 by specific pharmacological intervention could be a novel approach to modulate fibrosis progression or induce its reversal.


Detlef Schuppan - Advisory Committees or Review Panels: Aegerion, Eli Lilly, Gilead; Consulting: Boehringer-Ingelheim, Isis, Takeda; Grant/Research Support: Boehringer-Ingelheim

The following people have nothing to disclose: Shih-Yen Weng, Kornelius Padberg, Yong Ook Kim, Xiao-Yu Wang, Brombacher Frank


Whole Genome Study to Characterize Potential Determinants of Aplastic Anemia in HCV Patients Receiving Combination Anti-viral Therapy

Jacob George1, Alexander J. Thompson2, Steven L. Flamm3, Dongliang Ge4, Matthew Paulson4, Bittoo Kanwar4, Phil S. Pang4, Mani Subramanian4, John G. McHutchison4, David B. Goldstein5, Lisa M. Nyberg6, Thomas J. Urban5

1Starr Liver Unit, Westmead Millennium Institute, University of Sydney and Westmead Hospital, Sydney, NSW, Australia; 2Department of Gastroentrerology, St. Vincent's Hospital, University of Melbourne, Melbourne, NSW, Australia; 3Division of Gastroenterology and Hepatology, Northwestern Feinberg School of Medicine, Chicago, IL; 4Gilead Sciences, Inc., Foster City, CA; 5Center for Human Genome Variation, Duke University School of Medicine, Durham, NC; 6Kaiser Permanente, San Diego, CA

Background and Objectives: In Phase 2 trials of combination therapy for hepatitis C virus infection, 3 patients developed aplastic anemia. Two had been treated with peginterferon (PEG)+ribavirin (RBV)+tegobuvir+GS-9451 and 1 with PEG+RBV+ledipasvir+GS-9451. While bone marrow suppression is common with interferon therapy, aplastic anemia has been rare. Whole genome sequencing may identify rare genetic variants and reveal disease-causing variants in small samples. Methods: DNA extracted from 3 blood samples from studies GS-US-248-0121, GS-US-196-0123, and GS-US-1960140 were sequenced using the Illumina Whole Genome Sequencing (WGS) protocol and HiSeq 2000 sequencer. Average sequence coverage was 35X. Sequences were aligned to the most recent human reference genome (NCBI37/hg19). Entire genome sequences were compared with 425 whole genomes and 1054 whole exome sequenced population controls matched by ethnicity. The analysis focused on listing and prioritizing putatively functional rare (control MAF<0.001) variations predicted to alter amino acid sequence of protein coding genes carried by the aplastic anemia cases. A gene-wise collapsing test was performed to prioritize the genes enriched with such functional variants, and the Ingenuity Pathway Analysis (IPA) was used to analyze the prioritized list. PolyPhen-2 was used to predict the functional effects of genetic variants. Results: We identified 571 single nucleotide variants and 28 insertions/deletions affecting 573 putatively functional genes and carried by the cases only. Although none of the individual functional variants were shared by all 3 patients, we identified 24 common genes (Table 1) in which the 3 aplastic anemia cases harbor putatively functional variants. The top 2 functional categories revealed by IPA were developmental disorder (5 genes) and hematological disease (1 gene). One common gene SPTA1, spectrin, alpha, erythrocytic 1 (elliptocytosis 2) - was shared by the 2 categories. Two of the 3 aplastic anemia patients carry at least 1 heterozygous SPTA1 mutation; 1 carries a deleterious missense mutation (rs35237700, PolyPhen-2 score 1.000), and the other, 2 relatively benign mutations (rs150007668 and rs143459302, PolyPhen-2 score 0.249 and 0.001, respectively). Conclusions: We characterized the entire genomes of 3 patients who developed aplastic anemia during antiviral therapy, and identified 24 genes possibly associated with drug-induced aplastic anemia.

List of 24 genes with possible association with drug-induced aplastic anemia.



Jacob George - Advisory Committees or Review Panels: Roche, BMS, MSD, Gilead, Janssen

Alexander J. Thompson - Advisory Committees or Review Panels: Merck, Inc, Roche, Janssen (Johnson & Johnson), BMS, GSK Australia, Novartis, GILEAD Sciences, Inc; Consulting: GILEAD Sciences, Inc; Grant/Research Support: Merck, Inc, Roche, GILEAD Sciences, Inc; Speaking and Teaching: Merck, Inc, Roche, BMS

Steven L. Flamm - Advisory Committees or Review Panels: Merck, Gilead, Vertex, Merck, Gilead, Vertex, Merck, Gilead, Vertex, Merck, Gilead, Vertex; Grant/Research Support: Merck, Vertex, Gilead, Pfizer, Anadys, Achillion, Abbott, Tibotec, Merck, Vertex, Gilead, Pfizer, Anadys, Achillion, Abbott, Tibotec, Merck, Vertex, Gilead, Pfizer, Anadys, Achillion, Abbott, Tibotec, Merck, Vertex, Gilead, Pfizer, Anadys, Achillion, Abbott, Tibotec; Speaking and Teaching: Merck, Gilead, Vertex, Merck, Gilead, Vertex, Merck, Gilead, Vertex, Merck, Gilead, Vertex

Dongliang Ge - Employment: Gilead Sciences, Inc Matthew Paulson - Employment: Gilead Sciences Bittoo Kanwar - Employment: Gilead Sciences Phil S. Pang - Employment: Gilead Sciences Mani Subramanian - Employment: Gilead Sciences

John G. McHutchison - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

David B. Goldstein - Advisory Committees or Review Panels: Astra Zeneca, NIH, Biogen, Gordon Research Conference; Board Membership: Knome; Consulting: glaxo smithkline, Severe Adverse Events Consortium, Roche, Gilead Sciences, Inc, Scienta Advisors; Employment: Duke University; Grant/Research Support: UCB, NIH, Biogen, Henry M Jackson Foundation, SAIC, Inc, Bill & Melinda Gates Foundation, Eisai, Inc; Patent Held/Filed: patent IL28B findings, patent ITPA findings, Merck & Company; Speaking and Teaching: Current Biology magazine, Illumina, Regeneron, Dermatology Society; Stock Shareholder: Pfizer

Lisa M. Nyberg - Grant/Research Support: Merck, Roche/Genentech, Vertex, Bristol Myers Squibb, Gilead, Pharmasset, Abbott; Speaking and Teaching: Merck

Thomas J. Urban - Patent Held/Filed: Schering Plough