Figure 3. HCV dsRNA (NS-3’NTR)-Stimulated Macrophages or Supernatant (SN) From Macrophages Culture Suppresses HCV Replication in Huh7 Cells.

(A) Co-culture of HCV JFH-1-infected Huh7 cells with NS-3’NTR dsRNA-stimulated macrophages. Seven-day-cultured macrophages were plated in the low compartment of a 24-well plate and stimulated with NS-3’NTR dsRNA (1 μg/mL) for 16 hours, while HCV JFH-1-infected Huh7 cells (day 3 post infection) were plated in the upper compartment for co-culture for 48 hours. (B) Effect of SN of macrophages culture stimulated with NS-3’NTR dsRNA (1 μg/mL) on HCV replication in Huh7 cells. HCV JFH-1-infected Huh7 cells (day 3 post infection) were cultured in the presence or absence of SN of macrophages stimulated with NS-3’NTR dsRNA at indicated concentration for 48 hours. (C) Suppression of HCV RNA expression by macrophages SN under three different conditions. Huh7 cells were cultured in media conditioned with or without macrophages SN for either 24 hours prior to HCV infection, or simultaneously or 8 hours postinfection. The cells were then washed five times to remove input HCV after 6 hours incubation with HCV JFH1 and then cultured in the presence or absence of macrophages SN for 72 hours. Total cellular RNA extracted from hepatocytes was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. Data was expressed as HCV RNA levels relative (%) to control (LyoVec only, without co-culture, without stimulation or without SN treatment, which is defined as 100%). The results are mean ± SD of triplicate cultures, representative of three independent experiments (* P < 0.05, ** P < 0.01).