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, 82 (7), 3736-50

Immunomodulatory Properties of a Viral Homolog of Human interleukin-10 Expressed by Human Cytomegalovirus During the Latent Phase of Infection

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Immunomodulatory Properties of a Viral Homolog of Human interleukin-10 Expressed by Human Cytomegalovirus During the Latent Phase of Infection

C Jenkins et al. J Virol.

Abstract

Human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells, from which it can reactivate to cause significant disease in immunocompromised individuals. HCMV expresses a functional homolog of the immunosuppressive cytokine interleukin-10 (termed cmvIL-10), and alternate splicing of the cmvIL-10 transcript results in expression of a latency-associated cmvIL-10 transcript (LAcmvIL-10). To determine whether LAcmvIL-10 encodes immunosuppressive functions, recombinant LAcmvIL-10 protein was generated, and its impact on major histocompatibility complex class II (MHC-II) expression was examined on granulocyte macrophage progenitor cells (GM-Ps) and monocytes. LAcmvIL-10 (and cmvIL-10) downregulated MHC-II on the surfaces of both cell types. This downregulation was associated with a decrease in total MHC-II protein and transcription of components of the MHC-II biosynthesis pathway. Unlike cmvIL-10, LAcmvIL-10 did not trigger phosphorylation of Stat3, and its ability to downregulate MHC-II was not blocked by neutralizing antibodies to the human IL-10 receptor, suggesting that LAcmvIL-10 either does not engage the cellular IL-10 receptor or utilizes it in a different manner from cmvIL-10. The impact of LAcmvIL-10 on dendritic cell (DC) maturation was also assessed. In contrast to cmvIL-10, LAcmvIL-10 did not inhibit the expression of costimulatory molecules CD40, CD80, and CD86 and the maturation marker CD83 on DCs, nor did it inhibit proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6 and tumor necrosis factor alpha). Thus, LAcmvIL-10 retains some, but not all, of the immunosuppressive functions of cmvIL-10. As GM-Ps and monocytes support latent infection, expression of LAcmvIL-10 may enable HCMV to avoid immune recognition and clearance during latency.

Figures

FIG. 1.
FIG. 1.
MHC-II and CD14 expression on the surface of GM-Ps following treatment with purified LAcmvIL-10 and cmvIL-10. (A) Western blot of purified cmvIL-10 and purified LAcmvIL-10 separated by SDS-PAGE. Both proteins reacted with the polyclonal anti-cmvIL-10 antibody used to probe the blot with cmvIL-10 detected at 17.6 kDa and His-tagged LAcmvIL-10 detected at the predicted size of 16.8 kDa. Sizes of corresponding molecular mass markers are indicated in kilodaltons (kDa). (B) Histogram plot showing a decrease in surface MHC-II by LAcmvIL-10 and cmvIL-10 relative to mock protein preparation. Panels C to F show flow cytometric analyses of the expression of MHC-II (C and E) and CD14 (D and F) molecules on the surface of CD14+ GM-Ps treated with either bacterial cell-expressed and purified LAcmvIL-10 and cmvIL-10 (C and D) or conditioned medium (CM) from mammalian cells transfected with LAcmvIL-10 and cmvIL-10 expression vectors (E and F). The relative MFIs of surface MHC-II and CD14 molecules are shown with the means and standard errors calculated from four replicate experiments. Each data point represented the relative MFI of a given surface molecule (relative MFIM) on the various treatments of GM-Ps, calculated as follows: (MFIM on GM-Ps(treated)/MFIM on GM-Ps(mock)) × 100, where GM-Ps are treated with cmvIL-10, LAcmvIL-10, or mock preparation. Significant differences from the mock-treated control were determined using a one-tailed, paired Student's t test and are indicated as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
FIG. 2.
FIG. 2.
Ability of LAcmvIL-10 and cmvIL-10 to downregulate cell surface MHC-II on GM-Ps and monocytes in a dose-dependent manner. GM-Ps (A) and monocytes (B) were treated for 48 h with 10-fold decreasing concentrations of purified LAcmvIL-10, cmvIL-10, and mock protein preparations before being stained for CD14 and MHC-II and analyzed by flow cytometry. The relative MFIs of MHC-II surface molecules are shown with the means and standard errors calculated from at least three separate experiments. Each data point represents the relative MFI of a given surface molecule (relative MFIM) on the various treatments of GM-Ps, calculated as follows: (MFIM on GM-Ps(treated)/MFIM on GM-Ps(mock)) ×100, where GM-Ps are treated with cmvIL-10, LAcmvIL-10, or mock preparation. Significant differences from the mock-treated control were determined using a one-tailed, paired Student's t test and are indicated as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005, ****, P < 0.00005.
FIG. 3.
FIG. 3.
Expression of MHC-II protein by CD14+ monocytes treated with LAcmvIL-10 and cmvIL-10. (A) MHC-II (HLA-DRα) Western blotting performed on whole-cell lysates from monocytes treated with mock, cmvIL-10, or LA-cmvIL-10 protein preparations. Lysates from HLA-DR 9.22.3 cells were included as a negative control. The immunoblot was reprobed for the housekeeping gene GAPDH (bottom panel). (B) Graph depicting densitometer analysis of MHC-II bands from three replicate Western blotting experiments following normalization to GAPDH expression. Significant differences from the mock-treated control were determined using a one-tailed, paired Student's t test.*, P < 0.05.
FIG. 4.
FIG. 4.
Analysis of MHC-II (HLA-DR) protein localization in LAcmvIL-10- and cmvIL-10-treated CD14+ monocytes. Immunofluorescence staining and confocal microscopy visualization of MHC-II (HLA-DR; red staining) in monocytes treated with mock protein preparation (A), LAcmvIL-10 (C and E), and cmvIL-10 (G). Cells stained with isotype control antibody are shown in panel I. Corresponding phase-contrast images from each treatment are shown (B, D, F, H, and J). Arrows indicate the appearance of punctate cytoplasmic MHC-II staining in a small proportion of cells treated with LAcmvIL-10 and cmvIL-10. White bars indicate size.
FIG. 5.
FIG. 5.
Analysis of mRNA expression of components of the MHC-II biosynthesis pathway. Quantitative real-time RT-PCR analysis of HLA-DRα chain (A), HLA-DRβ chain (B), invariant chain (C), and CIITA (D) mRNA expression in monocytes treated for 48 h with mock, cmvIL-10, or LAcmvIL-10 protein preparations. Significant differences from the mock-treated control were determined using a one-tailed, paired Student's t test. **, P < 0.005.
FIG. 6.
FIG. 6.
Activation of Stat3 by cmvIL-10 but not LAcmvIL-10. (A) Primary human CD14+ monocytes were left untreated (Control) or treated with 10 ng/ml purified hIL-10, cmvIL-10, or LAcmvIL-10 protein preparations for 10 min. Cell lysates were then immunoblotted with polyclonal antiserum to detect phosphorylated or total unmodified Stat3 protein as indicated. (B) Monocytes were treated with 10 ng/ml purified cmvIL-10 or LAcmvIL-10 for increasing times and then immunoblotted as described above. (C) Monocytes were incubated with 10 ng/ml for each indicated cytokine or combination of cytokines for 10 min before lysis and blotting.
FIG. 7.
FIG. 7.
Capacity of LAcmvIL-10 and cmvIL-10 to downregulate MHC-II in the presence of neutralizing antibody to hIL-10R. Flow cytometric analysis of MHC-II (HLA-DR) expression on CD14+ GM-Ps following incubation without (A) or with (B) anti-hIL-10Rα/β neutralizing antibodies, prior to treatment with either LAcmvIL-10, cmvIL-10, or mock protein preparations. (C) The relative MFI values of surface MHC-II are shown with the means and standard errors calculated from four independent replicate experiments. Each data point represents the relative MFI of a given surface molecule (relative MFIM) on the various treatments of GM-Ps, calculated as follows: (MFIM on GM-Ps(treated)/MFIM on GM-Ps(mock)) ×100, where GM-Ps are treated with the cmvIL-10, LAcmvIL-10, or mock preparation. Significant differences from mock treatments were determined using a one-tailed, paired Student's t test. *, P < 0.05.
FIG. 8.
FIG. 8.
Effect of LAcmvIL-10 and cmvIL-10 on MDDC maturation following LPS stimulation. Flow cytometric analysis of the expression of CD40, CD80, CD83, CD86, and MHC-I molecules on the surface of LPS-induced DCs. The relative MFIs of surface molecules are shown with the means and standard errors calculated from four separate experiments. Each data point represents the relative MFI of a given surface molecule (relative MFIM) on the various treatments of LPS-induced DCs, calculated as follows: (MFIM on DCs(treated or immature)/MFIM on DCs(mock)) ×100, where DCs are immature or treated with the cmvIL-10, LAcmvIL-10, or mock preparation. Significant differences from the mock-treated control were determined using a one-tailed, paired Student's t test and are indicated as follows: **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005.
FIG. 9.
FIG. 9.
Impact of LAcmvIL-10 and cmvIL-10 on proinflammatory cytokine production by MDDCs following LPS stimulation. Multiplex ELISA-based analysis of the levels of cytokines production by DCs treated for 3 days with LPS in the presence of either purified cmvIL-10 protein or conditioned medium (CM) from HEK293 cells transfected with either cmvIL-10 or LAcmvIL-10 expression vectors. Uninduced DCs (i.e., immature DCs) and LPS-induced DCs treated with mock-conditioned medium are also included. The levels of IL-1α, IL-1β, IL-6, TNF-α, and GM-CSF in the supernatants of DCs relative to LPS-induced DCs treated with mock-conditioned medium are shown, with significant differences determined using a one-tailed, paired Student's t test and indicated as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005; *****, P < 0.000005; ******, P < 0.0000005; *******, P < 0.00000005.

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