HCMV-encoded UL128 enhances TNF-α and IL-6 expression and promotes PBMC proliferation through the MAPK/ERK pathway in vitro

Abstract

Cytomegalovirus (CMV) infection enhances expression of several cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-8, to the benefit of virus replication and dissemination. However, the stimulus for certain cytokine production remains unclear. CMV encodes a series of proteins that alter and/or mimic functions of leukocyte migration, activation, and cytokine responses. Our study revealed that human CMV (HCMV)-encoded UL128 protein, which contains signal peptides and has similar amino acid sequences to the CC chemokine, recruits monocytes as human β chemokine (microphage inflammatory protein 1α). Using RNA interference technology, we constructed an HCMV (UL128⁺/UL128⁻)-infected tissue cell (MRC-5) and peripheral blood mononuclear cell (PBMC) co-culture system. We measured 6 cytokine levels (IL-2, IL-4, IL-6, IL-10, TNF-α, and interferon-γ [IFN-γ]) in the supernatant, and found significantly elevated IL-6 and elevated TNF-α levels in the HCMV UL128⁺-infected group. Conversely, we observed decreased levels in the UL128-knockout supernatant. PBMCs presented with UL128 (50 ng/mL) demonstrated better cell viability than the UL128-absent group. Finally, the MAPK/ERK pathway was found to be involved in UL128 induction of cell proliferation. Selective induction of cytokine expression indicates that HCMV-encoded UL128 is a potent inducer of several inflammatory mediators.

Figures

FIG. 1.

Knockout of UL128 by short interference RNA (siRNA). A significant reduction of the UL128 transcript relative to a non-targeting control siRNA was detected in MRC-5 cells transfected with the above siRNA sequences shown in (a). (b) Fold differences and subsequent percent gene expression levels were calculated using the comparative CT (2−▵▵CT) method. Data were analyzed using one-way analysis of variance for comparison among groups, followed by pairwise comparisons using Tukey 95% confidence intervals.

FIG. 2.

The silver stain of isolated proteins. (M, marker; lane 1, the lysate of CHO transfected with empty vector pIRES-AcGFP; lane 2, elute from the lysate of CHO transfected with empty vector pIRES-AcGFP; lane 3, blank; lane 4, the lysate of CHO transfected with pIRES-AcGFP-UL128; lane 5, elute from the lysate of CHO transfected with pIRES-AcGFP-UL128).

FIG. 3.

Recombinant UL128 recruits peripheral blood mononuclear cells (PBMCs) in vitro. Migration assays were conducted by adding human PBMCs to the upper chamber of a 24-well plate with a pore size of 5 μm. The lower chamber was loaded with increasing concentrations of chemokine. The chemotactic index was calculated by the number of migrated cells divided by the random migration±standard deviation. Data were collected from three independent experiments (MIP-1α, macrophage inflammatory protein-1α).

FIG. 4.

UL128 treatment or human cytomegalovirus (HCMV) infection of peripheral blood mononuclear cells (PBMCs) increases levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). PBMCs were pre-treated with 50 ng/mL UL128 or elute from CHO transfected with empty vector pIRES-AcGFP. HCMV infection group: MRC-5 cells were cultured in 24-well plates and infected with HCMV. PBMCs were then co-cultured with MRC-5 cells. RNAi group: 100 nM of UL128 siRNA duplexes were transfected into MRC-5 cells, and seeded with HCMV, and lastly co-cultured with PBMCs. Blank: MRC-5 cells and a PBMC co-culture system without HCMV infection. Supernatants from these groups were collected at 18, 24, 48, 72, and 96 h, and cytokine levels were determined by flow cytometry. Data are representative of at least 3 independent experiments.

FIG. 5.

UL128 promotes PBMC proliferation through the MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase signaling) pathway. Peripheral blood mononuclear cells (PBMCs) cultured in the presence or absence of UL128 (50 ng/mL) for 72 h were analyzed by a MTT Cell Proliferation Assay Kit. A significant difference was found between the UL128-treated group and the negative control group (p<0.001). All the data shown represent the mean±standard deviation. Western blot analysis was done to detect ERK and pERK1/2 activation using antibody that specifically recognizes ERK and phosphorylated forms of ERK1/2. Lysates made from PBMCs (2×106cells/mL in the presence of 1% fetal bovine serum) were incubated for 30 and 60 min with UL128 (50 ng/mL). Endothelial growth factor (EGF) treatment of PBMCs and isolated PBMCs cultured for 30 or 60 min served as positive and negative controls, respectively (OD, optical density).