Figure 2. HCV stimulates IL-1β production upon uptake by macrophages.

(A) IL-1β mRNA expression (upper panel) and secreted IL-1β protein levels (lower panel) from primary monocyte-derived macrophages of healthy human donor blood. Cells were mock-treated or treated with infectious HCV supernatant (moi = 0.01 based on Huh7 focus forming units (ffu) or treated with 1 µg/ml of LPS and ATP 5 mM (LPS/ATP; positive control). (B) Intracellular cytokine staining of treated CD14+ cells recovered from saline washout of healthy donor liver. Cells were left untreated (unstim) or were cultured with conditioned media (mock, negative control), LPS (positive control) or treated with UV-inactivated HCV (moi = 0.01 based on Huh7 focus forming units (ffu)). Data are shown from one donor and are representative two experiments each of cells collected from two independent donors. In the analysis shown the frequency of IL-1β-expressing cells was: unstim, 2.7%; mock, 6.4%; LPS, 76.5%; UV-HCV. 67.6%. (C)–(I) Analysis of THP1 cells. (C) IL-1β mRNA expression post exposure to HCV. (D) IL-1β protein secretion after treatment with variable doses of HCV (moi = 0.001, 0.01 or 0.1 Huh7 ffu) or LPS/ATP at 1 µg/ml for 24 hr. (E) Immunoblot showing the kinetics of caspase-1 activation after HCV exposure. (F) Levels of secreted IL-1β over a time course after HCV exposure (moi = 0.01 based on Huh7 ffu). (G) IL-1β levels secreted 24 hr after exposure to (left to right) cell culture media, sucrose solution, sucrose-purified culture media, infectious HCV supernatant or sucrose-purified HCV virions. (H) IL-1β mRNA expression in pre-treated with DMSO (control), bafilomycin (2.5 uM) or cytochalasin D (10 µM) cells and exposed to media or infectious HCV supernatant (moi = 0.01 Huh7 ffu). (I) Levels of secreted IL-1β 24 hr post treatment with conditioned media alone (mock) or treatment with live infectious HCV (HCV, moi = 0.01) or UV-inactivated HCV (HCV-UV). *P = 0.0175 and ***P = 0.0005, by student t-test.