Volume 127, Issue 5, Pages 1513-1524 (November 2004) Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation  Angela Dolganiuc, Shilpa Oak, Karen Kodys, Douglas T. Golenbock, Robert W. Finberg, Evelyn Kurt-Jones, Gyongyi Szabo  Gastroenterology  Volume 127, Issue 5, Pages 1513-1524 (November 2004) DOI: 10.1053/j.gastro.2004.08.067 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Cell activation by HCV core and NS3 proteins requires TLR2 expression. (A) HEK/TLR2 and HEK/TLR4/MD-2 cells were stimulated with pLPS (0.1 μg/mL), PGN (10 μg/mL), zymozan (50 μg/mL), HCV proteins, or β-galactosidase (β-gal) as indicated, and IL-8 was determined in the supernatants after 12 hours by ELISA (n = 5). (B) Peritoneal macrophages from 5 mice per experimental groups from wild-type (TLR2+/+) and deficient (TLR2−/−) mice were stimulated overnight as detailed in Figure 1A. Mean ± SD TNF concentrations are shown. (C) HEK/TLR2 cells were stimulated with HCV proteins from different manufacturers and as described in Figure 1A, and IL-8 production was measured after overnight stimulation (n = 2). Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Activation of monocytes by HCV core and NS3 proteins. (A) Normal monocytes were stimulated with pLPS (0.1 μg/mL), PGN (10 μg/mL), HCV proteins as indicated, β-galactosamine (β-gal) control. Cytokine levels were determined after 12 hours stimulation. Mean ± SD from 5 experiments are shown. (B) Human monocytes were incubated with 10 μg/mL anti-TLR1 and/or anti-TLR2 antibody for 30 minutes at room temperature followed by stimulation with pLPS (0.1 μg/mL), PGN (10 μg/mL), core or NS3 (5 μg/mL), and PAM3 CSK4 (20 ng/mL) for 12 hours. Mean values ± SD from 2 experiments are shown. Asterisk indicates P value < .05 compared with controls determined by t test. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 MyD88 and IRAK are engaged upon cell stimulation with HCV core and NS3 proteins. (A) Peritoneal macrophages from wild-type (WT) or MyD88−/− mice were stimulated as indicated, and supernatants were analyzed 12 hours later for TNF-α production by ELISA. Mean values ± SD from 2 independent experiments are shown. Asterisk indicates P value < .05 compared with wild-type controls determined by t test. (B) CHO/TLR2 cells were stimulated as indicated for 20 minutes, and equal amounts of IRAK were precipitated and subjected to MAP kinase assay per stimulation group as detailed in the Materials and Methods section (n = 4). Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 HCV core and NS3 activate p38 and ERK pathways. (A) Human monocytes were stimulated with pLPS (0.1 μg/mL), PGN (10 μg/mL), core, NS3, or β-gal (5 μg/mL, each) for 20 minutes then lyzed on ice. Fifty micrograms of cellular proteins were tested with specific antibodies in Western blots. One representative experiment out of 3 with similar results is shown. (B) Human monocytes were preincubated with PD 98059 (50 μmol/L), SB203580 (10 μmol/L), or a corresponding dilution of DMSO vehicle control for 4 hours then stimulated as indicated in panel A, and TNF-α production was analyzed 12 hours later. Mean ± SD from 6 different donors are shown. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Human monocyte activation by HCV proteins leads to IκBα phosphorylation and NF-κB activation. Cells were stimulated as described in Figure 4 for 30 minutes for phospho-IκB or 60 minutes for total IκB and NF-κB. (A) Phospho- and total IκBα levels were determined by Western blots using 50 μg of protein. One experiment out of 3 with similar results is shown. (B and C) NF-κB activation was measured by EMSA using 5 μg of nuclear protein. One experiment (B) and the average densitometric units (C) of 5 are shown. (D) Monocytes were preincubated with MG132 (25 μmol/L) or a corresponding dilution of DMSO for 4 hours then stimulated as indicated, and TNF-α production was analyzed after 12 hours. Mean ± SD values from 6 different donors are shown. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 HCV core and NS3 activate JNK/SAPK pathway and AP-1. (A) Human monocytes were stimulated for 20 minutes as described in Figure 4. Phospho- and total JNK/SAPK and corresponding β-actin levels were determined by Western blot using 50 μg of total cellular protein. One experiment out of 3 with similar results is shown. (B and C) Cells were stimulated as indicated for 60 minutes, and nuclear proteins were analyzed for binding of 32P-labelled AP-1 nucleotide in EMSA. One blot (B) and densitometric analysis of 3 experiments (C) is shown. (D) Monocytes were preincubated with JNK II inhibitor (40 μmol/L) for 4 hours followed by stimulation as indicated for an additional 12 hours. TNF-α levels were determined in the supernatants by ELISA. Mean ± SD from 6 different donors are shown. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 Conformation and amino acid sequences affect the biologic activity of HCV proteins. (A) HCV proteins (5 μg/mL; core, aa 2–192 and NS3, aa 1450–1643) were heated to 95°C for 30 minutes, chilled on ice, and used to stimulate CHO/TLR2 cells for 60 minutes. Additional stimulations included PGN (10 μg/mL), pLPS (0.1 μg/mL), or medium control. Nuclear proteins were analyzed in EMSA. One representative blot from 2 experiments with similar results is shown. (B) HCV proteins (5 μg/mL) corresponding to the indicated amino acid sequences from the HCV polyprotein were used to stimulate HEK/TLR2 cells for 12 hours; supernatants were analyzed for IL-8 production by ELISA. Dotted line in nucleocapsid protein represents the missing parts of HCV polyprotein. Mean ± SD from 3 experiments are shown. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 8 TLR2 is required for activation but not for uptake of HCV core and NS3 proteins. (A) Human monocytes were incubated with HCV core and HCV NS3 for 1 hour or washed and incubated for an additional 3 hours (4 hours total) at 37°C. All cells were fixed with 4% paraformaldehyde, permeabilized were indicated and labeled with anti-HCV core or anti-HCV NS3 antibodies for 1 hour, followed by secondary PE-labeled antibodies. Cells were analyzed by flow cytometry. Representative histograms from 3 experiments with similar results are shown. (B) CHO and CHO/TLR2 cells were incubated with 10 μg/mL FITC-labeled HCV core and HCV NS3 or Dextran-FITC at 37°C or on ice for 1 hour then washed and immediately analyzed by flow cytometry (top). In the corresponding EMSA (bottom), CHO and CHO/TLR2 cells were stimulated as indicated, and 5 μg of nuclear proteins were analyzed for NF-κB activation. One representative blot out of 3 with similar results is shown. (C) CHO/TLR2 cells were preincubated with 5 μmol/L nocodazole (Noc) for 30 minutes and stimulated as indicated for 1 hour. Five micrograms of nuclear protein sample were analyzed in EMSA for NF-κB. One representative blot (bottom) and average densitometric values from 4 experiments are shown (top). (D) CHO/TLR2 were incubated with 10 μg/mL of HCV core-FITC or Dextran-FITC for 1 hour then permeabilized and incubated with anti-TLR2-PE antibodies and analyzed by confocal microscopy. Figure is representative of 2 experiments with similar results. Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 9 Increased TLR2-induced TNF-α production in monocytes from HCV-infected patients. Adherence isolated blood monocytes were stimulated for 12 hours as indicated, and TNF-α was measured by ELISA (*P < .05). Gastroenterology 2004 127, 1513-1524DOI: (10.1053/j.gastro.2004.08.067) Copyright © 2004 American Gastroenterological Association Terms and Conditions