Volume 138, Issue 1, Pages 305-314 (January 2010) Hepatitis C Virus Core Protein Subverts the Antiviral Activities of Human Kupffer Cells  Zhengkun Tu, Robert H. Pierce, Jonathan Kurtis, Yoshio Kuroki, I. Nicholas Crispe, Mark S. Orloff  Gastroenterology  Volume 138, Issue 1, Pages 305-314 (January 2010) DOI: 10.1053/j.gastro.2009.09.009 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 HCVc induces dose- and time-dependent expression of TNF-α, IL-1β, and IL-10. Human KC were incubated with recombinant HCVc. Cells and culture supernatants were collected at 16 hours. (A) Concentrations of IL-1β, TNF-α, and IL-10 were measured using a multiplexed cytokine bead assay. (B) Flow cytometry analysis of intracellular levels of IL-1β, TNF-α, and IL-10 in KC exposed to HCVc (10 μg/mL). Populations of cells incubated with HCVc are white and controls are black. Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 HCVc induces cytokine production from human KC and increases cytokine expression in response to agonists of TLR3 and TLR4 but not of TLR2. KC obtained from 5 live donor grafts were incubated with media alone (Med), HCVc (10 μg/mL), LTA (TLR2), poly-I:C (TLR3), LPS (TLR4), or combinations of a TLR agonist and HCVc. Supernatants were collected at 16 hours and analyzed by multiplex bead assays for IL-1β, TNF-α, and IL-10 levels. For each pair-wise comparison, the relevant P value is given. Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 HCVc-induced expression of TNF-α and IL-1β is blocked by soluble TLR2 (sTLR2). HCVc (1.0 μg/mL) was incubated in the presence of increasing concentrations of sTLR2; levels of TNF-α and IL-1β were quantified by intracellular cytokine staining. (A) A population of TNF-α-positive/IL-1β-positive KC appeared following stimulation with HCVc (1 μg/mL) in the absence of soluble TLR2, whereas addition of sTLR2 (1 μg/mL) almost completely inhibited this effect; representative contour plots are shown (n = 4) . LPS, a ligand for TLR4, was used as a specificity control. (B) The percent of positive KC was calculated based on the quadrant gates drawn as shown (open bars = HCVc stimulation, solid bars = LPS stimulation of KC). Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 HCVc induces expression of PD-L1 in human KC. KC were incubated with recombinant HCVc at concentrations ranging from 0 to 10 μg/mL and analyzed by flow cytometry after 4 hours, 8 hours, and 16 hours. (A) Expression of PD-l1 increased with time and level of HCVc (histograms shown representative of 3 experiments). (B) Mean fluorescence intensity (MFI) of PD-L1 expression, which increased over time and with concentration of HCVc. (C) In addition to HCVc, KC incubated with LTA, poly-I:C, or LPS stimulated a significant increase in PD-L1 by 16 hours compared with media alone. Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 HCVc suppresses poly-I:C-induced expression of TRAIL and type 1 IFN. (A) KC were incubated with recombinant HCVc (0, 1, and 10 μg/mL) and media alone (Med) or LTA, poly-I:C, or LPS; expression of TRAIL was analyzed at 16 hours by flow cytometry. Poly I:C was the only TLR ligand that induced TRAIL expression, whereas HCVc induced a dose-dependent decrease in poly-I:C-induced TRAIL expression (representative histograms are shown). (B) KC were incubated with media alone (Med), LTA, poly-I:C, LPS, and HCVc (10 μg/mL) or a combination of poly-I:C and HCVc (10 μg/mL). Only poly-I:C alone significantly increased TRAIL expression (shown as MFI), compared with Med; coincubation with HCVc completely inhibited the poly-I:C-induced increase in TRAIL. Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 PI3K activity is required for PD-L1 up-regulation by TLR ligands as well as TLR3-specific induction of TRAIL and type 1 IFNs. KC from 3 donors were incubated with media alone, TLR ligands (LTA, poly-I:C, or LPS), or HCVc in the presence of a specific PI3K inhibitor, LY294002. PD98059, a mitogen-activated kinase kinase inhibitor, was included as a control. (A) LY294002 inhibited TLR-mediated PD-L1- and poly-I:C-induced expression of TRAIL. (B) The TLR ligands and HCVc significantly increased the PD-L1 mean fluorescence intensity (MFI), compared with Med; this increase was completely inhibited by LY294002. (C) The MFI of TRAIL increased only in response to poly-I:C; this increase was completely inhibited by LY294002 (D and E). KC expression of IFN-β and IFN-α increased following stimulation with poly-I:C; this increase was blocked by LY294002. Gastroenterology 2010 138, 305-314DOI: (10.1053/j.gastro.2009.09.009) Copyright © 2010 AGA Institute Terms and Conditions