Human type 2 myeloid dendritic cells produce interferon-λ and amplify interferon-α in response to hepatitis C virus infection

Abstract

Background & aims: The type III interferons (IFN-λs: interleukin [IL]-28a, IL-28b, and IL-29) have important roles in hepatitis C virus (HCV) infection, but little is understood about what cells produce these cytokines or how production is activated. We investigated whether human immune cells recognize HCV-infected cells and respond by producing IFN-λ.

Methods: We cultured healthy human peripheral blood mononuclear cells (PBMCs) with different populations of immune cells and Japanese fulminant hepatitis-1 (JFH-1) HCV-infected Huh7.5 (cell culture-derived HCV particles [HCVcc]/Huh7.5) cells.

Results: Human PBMCs recognized HCVcc/Huh7.5 cells and responded by producing IFN-α, IFN-γ, and IFN-λ. A rare subset of myeloid dendritic cells (mDCs), which are blood DC antigen (BDCA)+ (also called mDC2 cells), were the major source of IL-28 and IL-29 production in response to HCVcc/Huh7.5 cells. Plasmacytoid DCs produced IFN-α, whereas natural killer and natural killer T cells were the main source of IFN-γ production in co-culture experiments. Of the endosomal Toll-like receptors (TLRs)3, 7, 8, and 9, only TLR3 or double-stranded HCV RNA induced production of IL-28 and IL-29 by mDC2s; endosomal maturation was required. Production of IFN-α and IFN-λ were linked-IFN-λ increased production of IFN-α by plasmacytoid DCs and IFN-α significantly increased production of IFN-λ.

Conclusions: mDC2s are a major source of IFN-λ production by PBMCs in response to HCVcc/Huh7.5 cells. mDC2s are activated through the TLR3 pathway, indicating that human DCs efficiently can initiate an immune response against HCV infection. IFN-λ therefore has an important role in HCV infection.

Figure 1. All three types of IFNs (IFN-α, IFN-γ and IFN-λs – IL-28 and IL-29) are produced from co-cultures of human PBMCs and HCV-infected cells

(A) Huh7.5 cells or HCVcc/Huh7.5 cells were co-cultured with PBMCs for 24 hours. IFNs in the supernatants were measured by ELISA (mean±SD; n=6). (B) HCVcc/Huh7.5 cells with different infection percentages (NS3 expression level) were co-cultured with human PBMCs for 24 hours. IFN-levels were measured by ELISA. Mean±SD are shown and every dot represents one data point. (C) Human PBMCs and hepatoma cells were collected before co-culture as 0h control or 4, 8 and 16 hours later. Total RNA was extracted from co-cultured cells and IFNs or MxA (MX1) expression were examined by real-time PCR, GAPDH as internal control (mean±SD; n=3). (D) Huh7.5 cells or HCVcc/Huh7.5 cells were co-cultured with PBMCs. Supernatants were collected 4, 8, 16 and 24 hours later and IFNs or IP-10 levels determined by ELISA (mean±SD; n=3-6).

Figure 2. IFN-α is produced by pDC, IFN-γ is produced by NK/NKT, while IFN-λ is produced by mDC2

(A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs, purified pDCs, PBMCs depleted of pDCs (PBMC-pDC), PBMCs depleted of NK/NKT (PBMC-CD56) or purified NK/NKT (CD56+) cells. IFNs in the supernatants were measured by ELISA after 24 hours (mean±SD; n=3-6 and (*) indicates p<0.05). (B) HCVcc/Huh7.5 cells were co-cultured with PBMCs, purified CD1c+ myeloid DCs (mDC1), monocyte derived DCs (MoDC), purified monocytes (CD14+ monocytes), CD16+ monocytes, platelet associated cells (CD61+), PBMCs depleted of granulocytes (PBMC-CD15), PBMCs depleted of monocytes (PBMCCD14), PBMCs depleted of pDC (PBMC-pDC), PBMCs depleted of platelet-associated cells (PBMCCD61) or PBMCs depleted of lymphocytes [PBMC-(T,B,NK,pDC)]. Co-cultured cells were preserved before co-culture as 0h control or collected after 4 hours. Total RNA was extracted from the cells and IL28 or IL29 expression examined by real-time PCR (mean±SD; n=2). (C) HCVcc/Huh7.5 cells were co-cultured with PBMCs, PBMCs depleted of mDC2 cells (PBMC-mDC2), or purified mDC2s. IL28 and IL29 expression from co-cultures was examined by real-time PCR (mean±SD; n=2).

Figure 3. mDC2s specifically express TLR3 and produce IFN-λs in response to HCV-dsRNA or poly I:C stimulation

(A-B) Human PBMC, purified pDCs, or mDC2s were co-cultured with HCVcc/Huh7.5 cells, or stimulated with different TLR ligands: TLR3 - Poly I:C (10μg/mL), TLR7 – Gardiquimod (1μg/mL), TLR7/8 - CL075 (2.5μg/mL) and TLR9 - CpG-A (2μM) for 24 hours. IFNs in the supernatants was measured by ELISA (mean±SD; n=3). (C) mRNA expression of TLRs (TLR3, 7, 8 and 9) and RLRs (DDX58 and IFIH1) was examined by real-time PCR from different immune subsets in PBMCs using GAPDH as internal control. Relative mRNA expressions are shown (mean±SD; n=3). (D) Gel electrophoresis of in vitro synthesized HCV ssRNA and dsRNA derived from the indicated HCV genome region. M: 1 kb DNA ladder (New England Biolabs Inc). (E-F) Isolated pDCs or mDC2s were stimulated by 10 μg/mL HCV-ssRNA or dsRNA mixed with lipofectamine 2000 for 24 hours. IFN levels are measured by ELISA (n=3). IFN-α production in pDCs was shown in (E), while IL-28 and IL-29 production in mDC2s was shown in (F).

Figure 4. Functional dichotomy of pDCs and mDC2s in IFN production

(A) Purified pDC or mDC2 populations were co-cultured with HCVcc/Huh7.5 cells and culture supernatants were collected at different time points for IFN-α and IL-29 measurement by ELISA (mean±SD; n=2). (B) PBMC-IFN production per cell was artificially set as 1 unit, then average IFN release from pDCs or mDC2s was calculated and compared (mean±SD; n=3). (C) Purified pDCs or mDC2s were co-cultured with HCVcc/Huh7.5 cells, or stimulated with CpG-A or Poly I:C respectively. 24 hours later, IL-28 or IL-29 in culture supernatants was measured by ELISA (mean±SD; n=3). (D) JFH-1 infected PHHs were collected 48h post infection for co-culture with human pDCs or mDC2s. Co-culture supernatants were collected at different timepoints and IL-28 or IL-29 levels were measured by ELISA. Representative data of one experiment was shown.

Figure 5. HCV-induced IFN-λ induction requires cell-to-cell contact and endosomal acidification

(A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs or purified mDC2s in a normal plate or a plate with a transwell insert. IL-29 in the supernatants was measured by ELISA after 24 hours (mean±SD; n=3), (B) Huh7.5 cells and HCV replicon cells – BB7 and FL cells were co-cultured with human PBMCs or purified mDC2s. IL-29 in culture supernatants was measured by ELISA (mean±SD; n=2), (C) HCVcc/Huh7.5 cells were co-cultured with purified mDC2 in the absence or presence of DMA, CCD or CLP. IL-29 production was measured by ELISA (mean±SD; n=2), (D) purified human mDC2s were co-cultured with HCVcc/Huh7.5 cells or stimulated with Poly I:C in the presence of chloroquine or bafilomycin as indicated. IL-29 production was measured by ELISA after 24 hours (mean±SD; n=2).

Figure 6. Cross-regulations exist between IFNs induction in co-cultures

(A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs in the absence or presence of recombinant IL-29 (25ng/mL). IFN-α and IFN-γ levels were measured by ELISA (mean±SD; n=3), (B) Human PBMCs or PBMC depleted of mDC2s (PBMC-mDC2) were co-cultured with HCVcc/Huh7.5 cells or stimulated with CpG-A. IFN-α production in the supernatants was measured by ELISA (mean±SD; n=3-4), (C) purified human pDCs were co-cultured with HCVcc/Huh7.5 cells or stimulated with CpG-A in the absence or presence of IL-29. IFN-α production was measured by ELISA (mean±SD; n=3), (D) total RNA was extracted from different immune subsets in human PBMCs. IL28RA expression was examined by real-time PCR and normalized to GAPDH (mean±SD; n=3), (E) Human PBMCs, Huh7.5 cells, HCVcc/Huh7.5 cells, co-culture of PBMCs and Huh7.5 cells or co-culture of PBMCs and HCVcc/Huh7.5 cells were preserved before culture as 0h control or collected after 4 hours culturing in the absence or presence of IFN-α-2a (100U/mL). IL28, IL29 and MX1 induction were measured be real-time PCR, one representative experiment of three is shown. (F) Human PBMCs or purified mDC2s were co-cultured with HCVcc/Huh7.5 cells or stimulated with Poly I:C in the absence or presence of IFN-α-2a. IL-29 production in the supernatants was measured by ELISA (mean±SD; n=3). (*) indicates p<0.05.

Figure 7

Genotype of rs12979860 does not correlate with IL-28 and IL-29 induction in human PBMCs by HCV-infected cells. (A) Human PBMCs were co-cultured with JFH-1 infected Huh7.5 cells or stimulated with PolyI:C (20μg/mL) or CpG-A (2μM) for 24 hours. Co-culture supernatants were collected and IL-28 or IL-29 levels determined by ELISA. Each dot represents one individual in the figures. (B) Circulating mDC1, mDC2 and pDC frequencies from normal control (NC) or naïve HCV patients (HCV) were determined by flow cytometry. (C) BDCA1 (CD1C), BDCA2 (CLEC4C), BDCD3 (THBD) and BDCA4 (NRP1) mRNA expression in human livers were determined by realtime PCR using GAPDH as internal control.