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. 2004 Aug;78(16):8720-31.
doi: 10.1128/JVI.78.16.8720-8731.2004.

Human cytomegalovirus-encoded interleukin-10 homolog inhibits maturation of dendritic cells and alters their functionality

W L William Chang  1 , Nicole Baumgarth, Dong Yu, Peter A Barry
Affiliations

Human cytomegalovirus-encoded interleukin-10 homolog inhibits maturation of dendritic cells and alters their functionality

W L William Chang et al. J Virol. 2004 Aug.

Abstract

Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

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Figures

FIG. 1.
FIG. 1.
Expression kinetics of HCMV ORF UL111A during viral replication. (A) UL111A is transcribed with late gene kinetics. MRC-5 cells were infected in triplicate with HCMV AD169 at an MOI of 1 and cultured in the presence (open symbols) or absence (filled symbols) of foscarnet (PFA; 167 μM) and ganciclovir (GCV; 4 μM). Supernatants were collected daily to quantify progeny virus titers by standard plaque assays (shown as lines), and total RNA was extracted for cDNA synthesis and quantitative real-time PCR analyses. The relative copy numbers of UL111A cDNA after normalization with GAPDH copy numbers are shown as bars. (B) Accumulation of cmvIL-10 protein in the supernatants of infected cultures. Cells were infected with HCMV strain AD169 (filled symbols) or Toledo (open symbols) at an MOI of 0.1. Supernatants were collected at indicated time points to measure virus titers (shown as lines) and cmvIL-10 protein concentrations (shown as bars). Shown are mean values ± standard deviations for triplicate cultures.
FIG. 2.
FIG. 2.
Genome structure and in vitro phenotype of HCMV AD169 mutant TS359. (A) Schematic diagram of HCMV genome structure with expansion of the UL111A ORF (exons and introns shown) of AD169 and the UL111A-mutated variant TS359. The UL111A ORF of TS359 is disrupted by the 3.66-kb transposon YD-Tn1721 insertion within its first exon (73). TRl, IRL, IRS, and TRS, terminal or internal inverted repeats flanking the Ul and Us components. (B) Multiple-step growth curves of AD169 (black symbols) and TS359 (gray symbols). Cells were infected with AD169 or TS359 at an MOI of 0.02, and supernatants were collected daily for measurement of progeny virus titers (shown as lines) and cmvIL-10 protein concentrations (shown as bars). ELISA data are shown as mean values ± standard deviations for triplicate cultures. (C) Immunoblot analysis for cmvIL-10 expression within HCMV-infected cells. Cell lysates were prepared after infection with AD169 or TS359 (MOI of 1) for 96 h. Immunoblots were probed for β-actin, HCMV gB, and cmvIL-10. The immunoblot of recombinant cmvIL-10 probed with anti-cmvIL-10 antibodies is also shown. Numbers at right are molecular masses in kilodaltons.
FIG. 3.
FIG. 3.
Inhibitory effects of HCMV-encoded IL-10 on DCs. iDC cultures were treated as indicated for 24 h, and their phenotypes were assessed by FACS and ELISA. Supernatants from AD169- and TS359-infected cultures were collected on day 7 postinoculation (Fig. 2B). Untreated DCs and DC cultures were incubated with conditioned medium from AD169- or TS359-infected cells (A), conditioned medium plus 1 ng of LPS/ml (B), conditioned medium from AD169-infected culture with 5 μg of anti-IL-10R1 MAb or IgG1 control/ml (C), or conditioned medium from TS359-infected culture with 1 ng of LPS/ml and indicated concentrations of recombinant cmvIL-10 (D). Surface expression of CD83 and HLA-DR on DCs was assessed by FACS to define immature (iDC; CD83− HLA-DRlow), intermediate (imDC; CD83− HLA-DRhigh), and mature (mDC; CD83+ HLA-DRhigh) DC populations, as indicated by boxes. Numbers represent frequencies of gated cells among the total DC population. Shown are 5% contour plots with outliers of cells after gating by forward scatter-side scatter to identify DCs by size. Cytokine data are expressed as the mean concentrations ± standard deviations of triplicate cultures. Results are representative of DCs from three different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 4.
FIG. 4.
Recombinant cmvIL-10 inhibits maturation of DCs and/or alters their cytokine expression profiles. (A) IL-10 inhibits LPS-stimulated but not CD40L-stimulated phenotypic maturation of DCs. Cells were unstimulated or stimulated with sCD40L (1 μg/ml) or LPS (at the indicated concentration) for 24 h in the absence (top panel) or presence of recombinant hIL-10 (second panel) or cmvIL-10 (bottom two panels). Results are representative of 10 separate experiments. (B) cmvIL-10 alters the IL-12 expression profiles of activated DCs. Cells were stimulated with 1 μg of sCD40L/ml or the indicated concentration of LPS and concomitantly treated with recombinant cmvIL-10 (0, 0.5, 5, and 50 ng/ml). Data are expressed as the mean concentrations ± standard deviations of triplicate cultures and are representative of five separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 relative to result with no cmvIL-10 treatment.
FIG. 5.
FIG. 5.
cmvIL-10 induces endogenous cIL-10 production. (A) cmvIL-10 enhances IL-10 secretion by activated DCs in a dose-dependent fashion. DCs treated with indicated concentrations of cmvIL-10 were stimulated for 24 h with sCD40L or indicated doses of LPS. Shown are mean concentrations from duplicate cultures representative of three separate experiments. (B) Shown are levels of cIL-10 produced by DCs from various donors (n = 9). Cells were stimulated with 10 ng of LPS/ml in the presence or absence of recombinant cmvIL-10 (50 ng/ml). Each symbol represents one individual donor. In all cases, cmvIL-10 stimulated the expression of cIL-10. The P value between untreated and cmvIL-10-treated groups is shown.
FIG. 6.
FIG. 6.
cmvIL-10 and hIL-10 share comparable potency in modulating the functionality of DCs. (A) IL-10 enhances phagocytic capacity of iDCs. DC cultures left untreated or treated with recombinant hIL-10 or vIL-10 (50 ng/ml) were analyzed by FACS for their ability to take up FITC-conjugated dextran (top panels) or FITC-conjugated beads (bottom panels) for assessment of endocytic and phagocytic capacity, respectively. Control panels on the left show untreated DCs with no FITC-dextran or FITC-conjugated beads added to the cultures. MFI, mean fluorescence intensity (FITC). Results are representative of two separate experiments. (B) IL-10 suppresses the induction of surface expression of costimulatory molecules on activated DCs. Cells were stimulated with 10 ng of LPS/ml in the presence (hIL-10, red open histogram; cmvIL-10, blue open histogram) or absence (gray shaded histogram) of recombinant IL-10 (5 ng/ml) for 24 h. Surface expression of indicated molecules on DCs was analyzed by FACS and presented as histograms after gating by forward scatter-side scatter-CD83-HLA-DR to identify mDC subsets. Expression levels of indicated molecules on the surface of iDCs are also shown (green open histogram). Isotype control staining is shown by dashed lines. (C) IL-10 alters cytokine secretion profiles of activated DCs. Levels of indicated proinflammatory cytokines in supernatants of DC cultures 24 h after LPS stimulation (10 ng/ml) and IL-10 treatment were determined by ELISA. Data are expressed as the mean cytokine concentrations ± standard deviations of triplicate cultures. Results are representative of eight separate experiments. **, P < 0.01.
FIG. 7.
FIG. 7.
Dual effects of cmvIL-10 on DC migration. (A) cmvIL-10 inhibits maturation of DCs and, thereby, inhibits their ability to migrate towards MIP-3β. Migration in response to MIP-3β (100 ng/ml) was assessed for DCs stimulated for 24 h with 1 ng of LPS/ml in the absence or presence of recombinant cmvIL-10 (50 ng/ml). Shown are 5% contour plots of input cells (top panels) and cells present in the bottom chamber 2 h after assay setup (bottom panels). Note that the only cells capable of migration were CD83+ HLA-DRhigh mDCs. Cells were gated by forward scatter-side scatter (FSC-SSC) to identify DCs by size. Results are representative of two separate experiments. (B to E) cmvIL-10 modulates the phenotype of migrating DCs. Cells stimulated with 10 ng of LPS/ml in the presence or absence of recombinant cmvIL-10 (50 ng/ml) for 24 h were used for chemotaxis assays. One representative experiment of three separate experiments is shown. (B) Input DCs were analyzed for surface expression of CD83-HLA-DR prior to chemotaxis. (C) Absolute numbers of CD83+ HLA-DRhigh cells in each well that migrated. Data are expressed as the mean numbers ± standard deviations of cells that migrated from triplicate assays. ***, P < 0.001. (D) Shown are the overlaid histograms for CCR7 expression on cmvIL-10-treated (open histogram) and untreated (gray shaded histogram) mDCs and the isotype control staining (dashed line). (E) The phenotypes of mDCs before (top panels) and after (bottom panels) cell migration towards MIP-3β were assessed by FACS. Surface expression of indicated molecules was analyzed by flow cytometry after gating by FSC-SSC-CD83-HLA-DR to identify mDC subsets. Results are presented as histograms, and numbers indicate mean fluorescence intensities (MFI) for the respective fluorochromes (gray number and shaded histogram, untreated; black number and open histogram, cmvIL-10 treated).
FIG. 8.
FIG. 8.
Activated mDCs treated with cmvIL-10 do not respond to further stimulation via CD40L with increased IL-12 production. DCs were stimulated with LPS (10 ng/ml) in the absence or presence of recombinant cmvIL-10 (50 ng/ml) for 12 h. Cells were collected, washed with PBS, and then stimulated with indicated concentrations of sCD40L for a further 24 h. Supernatants were harvested for measurement of IL-12 levels by ELISA. Results are expressed as the mean IL-12 concentrations ± standard deviations of triplicate cultures. *, P < 0.05; **, P < 0.01.
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