Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner
Fig 2
Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact.
(A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).