Role of A20 in interferon-α-mediated functional restoration of myeloid dendritic cells in patients with chronic hepatitis C

Abstract

Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon-α (IFN-α) -based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN-α treatment are unclear. The ubiquitin-editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN-α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV-infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN-α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN-α in vitro. Additionally, A20 expression by polyI:C-activated mDCs, with or without IFN-α treatment, negatively correlated with the expression of HLA-DR, CD86 and CCR7, and the secretion of interleukin-12 (IL-12), but positively associated with the production of IL-10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL-12 in mDCs of chronically HCV-infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.

Keywords: A20; chronic infection; hepatitis C virus; interferon-α; myeloid dendritic cells.

Figure 1

A20 expression increased in myeloid dendritic cells (mDCs) isolated from patients chronically infected with hepatitis C virus (HCV) compared with healthy subjects and is decreased in mDCs isolated from HCV patients receiving antiviral therapy. (a) Quantitative real-time PCR was used to detect A20 mRNA levels in mDCs isolated from treatment-naive patients (n = 31), patients receiving antiviral treatment (n = 16), patients achieving sustained virological responses (n = 10) and healthy subjects (n = 15). Each symbol represents an individual subject, and the horizontal bars represent the median values. (b, c) A20 expression in mDCs was assessed by flow cytometry. Corresponding changes in % gated and mean fluorescence intensity (MFI) are shown with isotype control in representative histograms (b). Summary data of corresponding changes in MFI from subjects from the four groups are shown by bar figure (c). *P < 0·05, **P < 0·05, ***P < 0·001, ns: not significant.

Figure 2

Interferon-α (IFN-α) down-regulates the expression of A20 mRNA in myeloid dendritic cells (mDCs) cultured in vitro. The mDCs purified from treatment-naive patients (n = 10) and healthy subjects (n = 10) were cultured in the presence of poly-IC, IFN-α, or IFN-α/poly-IC. A20 mRNA levels were detected by quantitative real-time PCR. (a) A20 mRNA expression in mDCs isolated from treatment-naive patients and healthy subjects and then treated for 12 hr with polyI:C (50 μg/ml). (b) A20 mRNA expression in mDCs isolated from healthy subjects and treatment-naive patients and then cultured with IFN-α (1000 U/ml) for 48 hr. (c) Myeloid DCs isolated from healthy subjects and treatment-naive patients and then cultured with IFN-α (1000 U/ml) for 48 hr. PolyI:C (50 μg/ml) was added to the culture for the final 12 hr. Each symbol represents an individual subject. *P < 0·05, **P < 0·01, ***P < 0·001, ns: not significant.

Figure 3

Interferon-α (IFN-α) down-regulates the expression of A20 and up-regulates the expression of HLA-DR, CD86 and CCR7 in myeloid dendritic cells (mDCs). The mDCs isolated from healthy subjects (n = 10) and treatment-naive patients (n = 10) were cultured in medium alone, or in medium containing polyI:C, IFN-α, or IFN-α/polyI:C. The expression of A20, HLA-DR, CD86 and CCR7 was then detected by flow cytometry. The percentage (mean ± SD) and the mean fluorescence intensity (MFI) of mDCs expressing A20 (a), HLA-DR (b), CD86 (c) or CCR7 (d) are shown. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 4

Myeloid dendritic cells (mDCs) show increased production of interleukin-12 (IL-12) and decreased production of IL-10 upon treatment with interferon-α (IFN-α). The mDCs isolated from healthy subjects (n = 10) and treatment-naive patients (n = 10) were cultured in medium alone or in medium containing polyI:C, IFN-α, or IFN-α/polyI:C. The levels of IL-12 (a) or IL-10 (b) secreted into the culture supernatants were measured by ELISA. Data represent the mean ± SD. *P < 0·05; ***P < 0·001.

Figure 5

A20 expression was negatively correlated with the expression of HLA-DR, CD86, CCR7 and interleukin-12 (IL-12), but positively correlated with IL-10 production. Immunophenotype molecules HLA-DR, CD86, CCR7 and cytokines IL-12, IL-10 in myeloid dendritic cells isolated from treatment-naive patients and activated with poly-I:C in the presence or absence of interferon-α were detected and their correlations with A20 were analysed by Spearman’s correlation analysis with two-tailed significance denoted in the upper right corner of each analysis. **P < 0·01, ***P < 0·001.

Figure 6

Silencing A20 rescues myeloid dendritic cell (mDC) interleukin-12 (IL-12) suppression and decreases IL-10 production in mDCs from treatment-naive patients. Myeloid DCs transfected with A20 small interfering RNA (siRNA) or control siRNA were subjected to RT-PCR and Western blot detection of A20 expression and ELISA of IL-12p70 and IL-10 secreted into the culture supernatant by mDCs. A20 siRNA inhibited hepatitis C virus (HCV) infection induced A20 expression by RT-PCR (a) and Western blot detection (b), a stronger silencing of A20 protein was achieved 48 hr and 72 hr after transfection. HCV-mediated IL-12 inhibition was partially restored (c) and the IL-10 production was decreased (d) in mDCs transfected with A20 siRNA. The data were reproducible in three repeated experiments.**P < 0·01, ***P < 0·001.