Gastroenterology

Gastroenterology

Volume 147, Issue 1, July 2014, Pages 209-220.e3
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Monocytes Activate Natural Killer Cells via Inflammasome-Induced Interleukin 18 in Response to Hepatitis C Virus Replication

https://doi.org/10.1053/j.gastro.2014.03.046Get rights and content

Background & Aims

Production of interferon (IFN)-γ by natural killer (NK) cells is attenuated during chronic infection with hepatitis C virus (HCV). We investigated whether this is due to intrinsic or extrinsic mechanisms of NK cells.

Methods

Peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic HCV infection or uninfected blood donors (controls); NK cells and monocytes were isolated or eliminated. We cultured hepatoma cells that express luciferase-tagged subgenomic HCV replicons (Huh7/HCV replicon cells) or their HCV-negative counterparts (Huh7) with NK cells in the presence or absence of other populations of PBMCs. Antiviral activity, cytotoxicity, and cytokine production were assessed.

Results

NK cells produced greater amounts of IFN-γ when PBMC were cocultured with Huh7/HCV replicon cells than with Huh7 cells; NK cells and PBMCs from controls suppressed HCV replication to a greater extent than those from patients with chronic HCV infection. This antiviral effect was predominantly mediated by tumor necrosis factor (TNF)-α and IFN-γ. The antiviral activity of NK cells and their production of IFN-γ were reduced when they were used in coculture alone (rather than with PBMC), or after depletion of CD14+ monocytes, after knockdown of the inflammasome in monocytes, or after neutralization of interleukin-18, which is regulated by the inflammasome. These findings indicate a role for monocytes in NK cell activation. Compared with control monocytes, monocytes from patients with chronic HCV infection had reduced TNF-α–mediated (direct) and reduced NK cell–mediated (indirect) antiviral effects. Control monocytes increased the antiviral effects of NK cells from patients with chronic HCV infection and their production of IFN-γ.

Conclusions

Monocytes sense cells that contain replicating HCV and respond by producing interleukin-18 via the inflammasome and by activating NK cells. Patients with chronic HCV infection have reduced monocyte function, attenuating NK cell IFN-γ–mediated responses.

Section snippets

Isolation of PBMCs and PBMC Subfractions

Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats or heparin-anticoagulated blood from chronically HCV-infected (Supplementary Table 1) or uninfected subjects on Ficoll-Histopaque (Mediatech, Manassas, VA) and washed 3 times with phosphate-buffered saline (Mediatech). Monocytes or pDCs were depleted with CD14+ or CD304 microbeads (Miltenyi Biotec, Auburn, CA), respectively. Alternatively, NK cells and monocytes were negatively selected with NK and Pan-Monocyte Isolation

PBMCs Exert a Greater Cytokine-Mediated Antiviral Effect Than Isolated NK Cells in Cocultures With Huh7/HCV Replicon Cells

To assess the antiviral activity of NK cells, we cocultured PBMCs from healthy blood donors with Huh7 cells that were stably transfected with a luciferase-tagged subgenomic HCV replicon (Huh7/HCV replicon cells). Whereas PBMCs down-regulated HCV replication by 74.9%, isolated NK cells decreased replication by only 20.8% (P < .0001) at an E/T ratio adjusted to the frequency of NK cells in PBMCs (Figure 1A). Down-regulation of HCV replication was associated with minimal cytotoxicity (Figure 1B)

Discussion

In this study, we used an in vitro model of HCV replication to investigate mechanisms of NK cell activation and antiviral function. We showed that NK cells from healthy subjects sense HCV-replicating hepatoma cells even though no HCV virions are produced. Similar to CD8 T cells in this model,15 NK cells mediate their antiviral effect predominantly through cytokine production rather than cytotoxicity. Moreover, similar to CD8 T cells from patients with chronic HCV infection,20, 21 NK cells from

Acknowledgments

The authors thank Xenia Chepa-Lotrea for technical support and Nevitt Morris for clinical support.

References (29)

  • B. Oliviero et al.

    Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections

    Gastroenterology

    (2009)
  • B. Rehermann

    Pathogenesis of chronic viral hepatitis: Differential roles of T cells and NK cells

    Nat Med

    (2013)
  • A.I. Su et al.

    Genomic analysis of the host response to hepatitis C virus infection

    Proc Natl Acad Sci U S A

    (2002)
  • E.C. Shin et al.

    Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection

    J Clin Invest

    (2006)
  • Cited by (0)

    Author names in bold designate shared co-first authors.

    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health intramural research program. J.M.W. was supported by grant We-4675/1-1 from the Deutsche Forschungsgemeinschaft (Bonn, Germany).

    Authors share co-first authorship.

    View full text