Original ResearchFull Report: Basic and Translational—LiverMonocytes Activate Natural Killer Cells via Inflammasome-Induced Interleukin 18 in Response to Hepatitis C Virus Replication
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.
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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).
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Authors share co-first authorship.