Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner

Abstract

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release.

Fig 1. Flt3-L derived DC, but not GM-CSF mDC, are activated after coculture with HCV transfected hepatoma cells and produce type I and type III IFN.

Huh7.5 cells were transfected with HCV subgenomic replicon (SGR) RNA or HCV full length (Jc1) RNA and incubated for 72 h. Murine Flt3-L derived DC or GM-CSF derived DC were cocultured with mock or HCV RNA transfected hepatoma cells or stimulated with VSV-M2 at a MOI 1 for 18 h and cells were analyzed by flow cytometry or ELISA. (A) Exemplary gating on SiglecH⁺ CD11c⁺ Flt3-L DC and analysis of the CD69 expression. (B) Exemplary gating on CD11c⁺ CD11b⁺ GM-CSF DC and analysis of the CD69 expression. (C) Quantification of the mean fluorescent intensity (MFI) of CD69 expression by Flt3-L DC or GM-CSF DC after coculture and stimulation (n = 3–4). Analysis of IFN-α (D), IFN-β (E) and IFN-λ (F) in cell-free supernatants of Flt3-L derived DC cultures by ELISA. Analysis of IFN-α (G), IFN-β (H) and IFN-λ (I) in cell-free supernatants of GM-CSF derived DC cultures (n = 3–6). 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; 2-way ANOVA, means + SD; n.s. not significant).

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).

Fig 3. Type I IFN production is dependent on IFNAR and TRIF signaling.

Flt3-L DC were generated from (A) C57BL/6, (B) IFNAR^-/-, (C) IL28R^-/-, (D) CARDIF^-/-, (E) MyD88^-/- and (F) TRIF^-/- mice and cocultured with mock or HCV transfected cells or stimulated with VSV-M2 at a MOI 1. After 18 h, IFN-α was quantified in cell-free supernatant by ELISA (n = 3–8). 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, means + SD; n.s. not significant).

Fig 4. In the Flt3-L derived DC cultures pDC, CD11b-like DC and CD8α-like DC show enhanced CD69 expression after coculture with HCV transfected hepatoma cells.

Flt3-L derived DC were co-cultivated with HCV transfected hepatoma cells or stimulated with VSV-M2 at a MOI 1 for 18 h and analyzed by flow cytometry. Cells were gated on (A) SiglecH⁺ CD11c⁺ pDC, (B) SIRPα⁺ CD11b⁺ CD11b-like DC or (C) Clec9A⁺ CD24⁺ CD8α-like DC and analyzed for the up-regulation of CD69 expression. (D) Quantification of the mean fluorescent intensity (MFI) of CD69 expression by Flt3-L derived pDC, CD11b-like DC and CD8α-like DC (n = 3) (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; 2-way ANOVA, means + SD; n.s. not significant).

Fig 5. HCV replication stimulates Flt3-L CD8α-like DC to produce type I IFN.

Flt3-L DC cultures were FACS sorted into pDC, CD11b-like DC and CD8α-like DC or the respective residual cells and co-cultured with mock or HCV transfected cells or stimulated with VSV-M2 at a MOI 1 for 18 h and the IFN-α amount in the supernatant measured by ELISA. Coculture with Flt3-L DC (A), sorted Flt3-L pDC (B), sorted CD11b-like DC (C), sorted CD8α-like DC (D), Flt3-L non-pDC (residual cells from pDC sort) (E), Flt3-L non-CD11b-like DC (residual cells from CD11b DC sort) (F) and Flt3-L non-CD8α-like DC (residual cells from CD8α-like DC sort) (G) (n = 4). 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, means + SD; n.s. not significant).

Fig 6. HCV-replicating cells induce IFN-β induction in vivo.

(A) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ mock transfected (left flank) or HCV subgenomic RNA transfected (SGR2; right flank) Huh7.5 cells. At the indicated time points post s.c. injection, luciferin was injected i.v. and luciferase activity was measured using the IVIS Spectrum in vivo imaging system. (B) Quantification of luciferase activity. Each symbol represents a region of interest (ROI) analysis of an individual animal (n = 6). (C) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ pUCΔGDD (ΔGDD) transfected or HCV SGR RNA transfected Huh7.5 cells treated with or without the HCV protease inhibitor telaprevir. At the indicated time points post s.c. injection, luciferin was injected i.v., the luciferase activity measured using the IVIS Spectrum in vivo imaging system and the signal quantified. Each symbol represents an individual animal (n = 3–4). (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; 2-way ANOVA, means + SD; n.s. not significant).