cmvIL-10 activates Stat3 but not Stat1 in monocytes. A. Cell lysates from primary human CD14⁺ monocytes treated with 10 ng/ml recombinant hIL-10 or cmvIL-10 (R&D Systems) for 10 min were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with polyclonal antiserum (Cell Signaling) to detect phosphorylated or total unmodified Stat protein as indicated. Monocytes were isolated from buffy coats of healthy human donors by Ficoll density gradient centrifugation and magnetic separation with anti-CD14 microbeads (Miltenyi). B. Cells were pretreated with 10 μM JAK1 inhibitor {2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one, dissolved in dimethyl sulfoxide; Calbiochem} or MEK1 inhibitor (U0126; Cell Signaling) for 1 hour prior to treatment with 10 ng/ml cmvIL-10, and then lysates were immunoblotted. The total protein in each lysate was quantified (Bio-Rad protein assay kit), and 10 μg total protein was loaded in each well. Results are representative of experiments performed on cells from three different donors.

Inhibition of TNF-α does not require JAK1 activity. Monocytes were incubated with JAK1 inhibitor for 1 h prior to treatment with 1 ng/ml LPS in the presence or absence of 10 ng/ml cmvIL-10. After 18 h, supernatants were collected and TNF-α levels were detected via sandwich ELISA (R&D Systems). Gray bars, LPS treatment only; black bars, LPS plus cmvIL-10. Error bars show standard deviations. Statistical analysis was performed using Student's t test, and differences in TNF-α production between cmv-IL-10-treated and untreated monocytes were significant regardless of inhibitor dose. *, P < 0.01. Results are representative of experiments performed on cells from five different donors in 96-well plates with 5 × 10⁴ cells per well.

PI3K plays a role in cmvIL-10-mediated cytokine suppression. (A) LPS-stimulated monocytes (1 ng/ml) were incubated with a panel of different commercially available signal transduction inhibitors [JAK3 inhibitor 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (Calbiochem), MEK1 inhibitor U0126 and PI3K inhibitor LY249002 (Cell Signaling), or wortmannin and p38 mitogen-activated protein kinase inhibitor SB203580 (Sigma)] in the presence or absence of 10 ng/ml cmvIL-10. Trypan blue analysis confirmed cells from each treatment were viable and then supernatants were analyzed for TNF-α levels by ELISA after 18 h. Gray bars, LPS treatment only; black bars, LPS plus cmvIL-10. Statistical analysis showed that TNF-α levels increased significantly in the presence of cmvIL-10 only with treatment with PI3K inhibitors LY249002 and wortmannin. *, P < 0.01, Student's t test. Results are representative of experiments performed on cells from five different donors. (B) Monocytes were incubated in the presence of the JAK1 inhibitor (circle), the PI3K inhibitor (LY249002; square), or both inhibitors (triangle) for 1 hour prior to LPS stimulation in the presence of 10 ng/ml cmvIL-10 and then TNF-α levels were measured by ELISA. A single black diamond indicates the level of TNF-α in the presence of LPS only. Results are representative of experiments performed on cells from three different donors. Error bars represent standard deviations.

cmvIL-10 induces PI3K-dependent phosphorylation of Akt and Stat3 (S7272). Human monocytes were treated with 50 μM JAK1 or PI3K inhibitor (LY249002) for 1 h prior to incubation with 10 ng/ml cmvIL-10 and then lysates were immunoblotted with polyclonal antiserum (Cell Signaling) to phosphorylated or total unmodified Stat3 or Akt protein. Results are representative of experiments performed on cells from three different donors.