Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

Abstract

The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14(+)monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression.

Importance: Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

FIG 1

Human CD14⁺ monocytes exhibit increased hIL-10 protein secretion in response to cmvIL-10. (A) ELISA-based quantitation of hIL-10 in the supernatants of cultures of primary human peripheral blood-derived CD14⁺ monocytes treated for 24 h with cmvIL-10 (100 ng/ml) or with phosphate-buffered saline (control). (B) Dose-dependent upregulation of hIL-10 secretion by CD14⁺ monocytes cultured with increasing concentrations of cmvIL-10 or with phosphate-buffered saline (control). The number (N) of independent biological replicate experiments is shown. Error bars indicate the standard errors of the means. Significant differences between results for the test samples and those of the control were determined using a two-tailed, paired Student's t test and are denoted by asterisks (*, P < 0.05; ****, P < 0.0001).

FIG 2

cmvIL-10 is biologically active in the absence of hIL-10. (A) Flow cytometry-based assessment of cell surface CD163 by CD14⁺ monocytes pretreated with hIL-10 neutralizing antibody or its isotype control antibody for 2 h prior to incubation for 24 h with cmvIL-10, hIL-10, or phosphate-buffered saline (Control). Open histograms depict CD163 after staining with a CD163-specific antibody, and filled histograms depict CD163 expression after staining with an isotype control antibody. (B) Graph depicting the mean fluorescence intensity (MFI) of cell surface CD163 by CD14⁺ monocytes pretreated with hIL-10 neutralizing antibody or isotype control antibody followed by hIL-10 or cmvIL-10 treatment. Error bars indicate the standard errors of the means from three independent biological replicate experiments. Significant differences between results for the test samples and those with the control were determined using a two-tailed, paired Student's t test and are denoted by asterisks (*, P < 0.05).

FIG 3

cmvIL-10 upregulates transcription of hIL-10 and TPL2 but not DUSP1. CD14⁺ monocytes were treated with cmvIL-10 or with phosphate-buffered saline (Control), and qRT-PCR was used to quantify the fold change in mRNA expression of hIL-10, TPL2, and DUSP1, determined relative to the levels with treatment with phosphate-buffered saline (Control). The number (N) of independent biological replicate experiments is shown. Error bars indicate the standard errors of the means. Significant differences between results for the test samples and those of the control were determined using a two-tailed, paired Student's t test and are denoted by asterisks (*, P < 0.05).

FIG 4

cmvIL-10 requires binding to hIL-10R and functional HO-1 for induction of hIL-10 protein secretion. (A) hIL-10 protein secretion determined by ELISA in supernatants from CD14⁺ monocytes pretreated with the HO-1 inhibitor ZnPP or the HO-1 inducer CoPP for 2 h prior to incubation for 24 h with cmvIL-10 or with phosphate-buffered saline (Control). (B) Graph depicting the amount of secreted hIL-10 determined by ELISA from CD14⁺ monocyte cultures pretreated with hIL-10R neutralizing antibody or its isotype antibody for 2 h prior to 24 h of treatment with cmvIL-10. (C) Representative flow cytometry histograms showing expression of intracellular HO-1 in CD14⁺ monocytes pretreated with neutralizing antibody to hIL-10R or its isotype control antibody. Open histograms depict HO-1 expression after treatment with cmvIL-10. Filled histograms depict results with phosphate-buffered saline (Control), and dotted histograms depict results after staining with an isotype control antibody. (D) Graph depicting the mean fluorescence intensity (MFI) of HO-1 determined by intracellular flow cytometry from CD14⁺ monocyte cultures pretreated with hIL-10R neutralizing antibody or its isotype antibody The number (N) of independent biological replicate experiments is shown. Error bars indicate the standard errors of the means. Significant differences between results for the test samples and those with the control were determined using a two-tailed, paired Student's t test and are denoted by asterisks (*, P < 0.05).

FIG 5

PI3K and STAT3 are required for cmvIL-10-mediated induction of HO-1 and hIL-10. CD14⁺ monocytes were pretreated with PI3K inhibitor (LY-294002; 50 μM) and STAT3 inhibitor (S3I-201; 50 μM) or with DMSO (Control) for 4 h prior to incubation for 18 h with cmvIL-10. (A) Histograms showing HO-1 and GAPDH levels by intracellular flow cytometry. Open histograms depict results for samples treated with cmvIL-10, and filled histograms depict results for samples treated with DMSO (Control). Dotted-line histograms depict results for samples treated with an isotype control antibody. (B) Graph depicting the intracellular HO-1 mean fluorescence intensity (MFI) values in monocyte cultures treated with cmvIL-10, with or without inhibitors, relative to those of the control. (C) Graph depicting the levels of secreted hIL-10 in monocyte cultures treated with cmvIL-10, with or without inhibitors, relative to those of the control. The number (N) of independent biological replicate experiments is shown. Error bars indicate the standard errors of the means. Significant differences between results for test samples and those with the DMSO control were determined using a two-tailed, paired Student's t test and are denoted by asterisks (*, P < 0.05).