Human cytomegalovirus suppresses type I interferon secretion by plasmacytoid dendritic cells through its interleukin 10 homolog

Abstract

Type I interferons (IFNs) are innate cytokines with potent antiviral and immunoregulatory activities. It remains unclear how human cytomegalovirus (HCMV) can establish persistence in the face of these strongly antagonistic cytokines. In this study, we confirm that IFN-alpha efficiently suppresses the penetration of HCMV into susceptible cells, including monocytes, the major cell population in peripheral blood that is highly susceptible to HCMV infection. We further demonstrate that the HCMV-derived interleukin 10 (IL-10) homolog functions similar to cellular IL-10 and broadly inhibits TLR-induced transcriptional activation of IFN-alpha/beta genes in plasmacytoid dendritic cells (PDCs), a major type I IFN-producer in vivo that is highly resistant to HCMV infection in vitro. These results suggest that HCMV subverts innate immunity by suppressing type I IFN production of PDCs during primary viral infection via its IL-10 homolog.

Fig. 1. HCMV targets monocytes and myeloid DCs in peripheral blood

Freshly isolated blood CD14⁺ monocytes (left panels), CD11c⁺ DCs (middle panels) and CD123⁺ PDCs (right panels) were infected for 36 h with GFP-recombinant HCMV at MOI’s of 1 and 5. The expression of indicated surface markers and of GFP identifying HCMV-infected cells was assessed by FACS. Shown are 5% contour plots with outliers of cells after gating leukocytes via FSC–SSC and CD14⁺to identify monocytes, FSC–SSC and CD11c⁺ to identify CD11c⁺ DCs, and FSC–SSC and CD11c^–/CD123⁺ to identify PDCs. The percentage of GFP⁺ cells, among the identified cell populations is shown in each plot, indicated by a box. Numbers are presented as the mean values ± SD of data from 3–4 individual donors.

Fig. 2. IFN-α reduces the susceptibility of various cell types to HCMV infection

Monolayer cultures of MRC-5 fibroblasts, ARPE-19 epithelial cells and freshly isolated CD14⁺ monocytes were pre-treated with indicated doses of IFN-α before HCMV infection. Data represent mean frequencies of infected cells ± SD from triplicate or quadruplicate cultures, as determined by FACS. Statistical comparisons to control samples without IFN-α treatment were performed using one-way ANOVA followed by Dunnett’s post tests (**p < 0.01). The results of monocytes are representative of experiments from two individual donors that gave similar results.

Fig. 3. cmvIL-10 inhibits IFN-α production by PBMCs

Freshly isolated PBMCs were treated with CpG and conditioned medium from HCMV-infected cultures (A), CpG or heat-inactivated influenza virus in the presence or absence of indicated concentrations hIL-10 or cmvIL-10 (ng/ml) (B). The supernatants were collected after stimulation for 24 h and IFN-α concentrations were determined by ELISA. Data represent mean values ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post tests (***p < 0.001) (A) or Dunnett’s post tests in comparison with the control samples without IL-10 treatment (**p < 0.01) (B). Shown are representative of data from one of two individual donors.

Fig. 4. cmvIL-10 inhibits IFN-expression but not maturation of PDCs

Freshly isolated PDCs were left untreated or stimulated with CpG in the presence or absence of 5 ng/ml hIL-10 or cmvIL-10 for 18 h. The surface expression of indicated molecules on PDCs was assessed by FACS and is presented with overlaid histograms. Shown are representative data from one of five individual donors that gave similar results (A). IL-10 significantly reduces type I IFN production by CpG-activated PDCs. IFN-α (upper panel, n = 5) or IFN-β (lower panel, n = 3) protein concentrations in supernatants were measured by ELISA. Each dot represents the relative concentrations (%) in the cultures compared to CpG-only stimulation from individual donors. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post tests (***p < 0.001) (B).

Fig. 5. Effects of cmvIL-10 on the transcription of type I IFN genes in activated PDCs

IL-10 suppresses IFN-β gene expression induced through TLR signals. Shown are fold-changes in gene expression compared to the untreated cells after normalization (A). Impacts of hIL-10 and cmvIL-10 on the expression of all IFN-α subtypes in CpG-activated PDCs. Shown are the relative mRNA copy numbers after normalization to the expression of an internal control gene. Data presented here are the mean values of the results from 2 individual donors (B). These data are part of a large analysis on IFN-α subset responses of PDCs that we have published previously (Szubin et al.), in which we did not distinguish between the effects of hIL-10 and cmvIL-10.