Fig 2: IL-27 increases the expression of p47phox in macrophages.(a) M-Mac were transfected with either control si-RNA (si-Ctlr) or si-RNA targeting p47phox (si-p47) followed by treatment with or without 100 ng/ml IL-27 for 48 h at 37 °C. As a control, un-transfected cells (Mock) were treated with IL-27. 48 h after transfection, whole cell lysates were prepared and western blotting was performed using anti-p47phox or anti-ß-actin antibodies. (b) si-Ctlr, si-p47 or Mock-transfected cells were treated with IL-27 for 48 h at 37 °C and superoxide production was analyzed by measuring H2O2 within culture supernatants. Data shown represent means ± SD of triplicate samples. (c) Macrophages were transfected with an empty or a p47phox expression vector for 6 h, and then the expression levels of p47phox were determined by Western blot. (d) H2O2 induction by PMA stimulation was detected by ROS assays following transfection of either the empty or p47phox expression vector into macrophages. Data shown represent means ± SD of triplicate samples. (e,f) Monocytes from a healthy control or a CGD patient lacking the expression of p47phox were differentiated into macrophages. Cells were then incubated with or without IL-27 for 48 h at 37 °C. The resulting cells were then stimulated with or without 100 ng/ml PMA for 30 min and ROS activity was measured by detection of H2O2 in culture supernatants. Data shown represent means ± SDs of triplicate samples from three independent studies. *P < 0.01.

Fig 3: IL-27 enhances superoxide production in GM-CSF-induced macrophages and iDC.(a) Monocytes were differentiated in the presence of GM-CSF and IL-4 into iDCs, GM-CSF alone into GM-Mac, or M-CSF alone into M-Mac from the same lot of monocytes. Differentiated cells were subsequently cultured for 48 h at 37 °C either in the absence or presence of 100 ng/ml IL-27. Using gene specific probes, mRNA expression levels of p47phox and gp91phox were quantified by RT-PCR. Values were normalized to GAPDH levels in untreated cells. Data shown represent means ± SE of three independent studies. (b) Whole cell lysates from iDCs, GM-Mac, and M-Mac with or without stimulation by IL-27 were analyzed by western blotting for p47phox, gp91phox, and ß-actin expression as an internal control. (c) GM-Mac, M-Mac and iDC were cultured in the presence or absence of IL-27 and then treated with or without 100 ng/ml of PMA for 30 minutes. ROS activity was measured in the culture supernatants. Data shown represent means ± SE of three independent studies. **P < 0.05.

Fig 4: JAK inhibitors suppress the IL-27 induction of p47phox expression and PMA-induced superoxide production.M-Mac were treated with 5 µM of Tofacitinib (Tof) or 1 µM of Ruxolitinib (Rux) for 1 h at 37 °C, and then cells were cultured for 48 h in the presence or absence of 100 ng/ml of IL-27. (a) Total cellular RNA was extracted from the cells and then p47phox mRNA expression was detected by real time PCR as described in the experimental procedures. Data show representative means ± SDs of 3 independent experiments. (b) Total cell lysate was prepared with RIPA buffer and then western blotting was performed using anti-p47phox antibody. Anti-ß Actin antibody was used as a loading control. (c) PMA-induced superoxide production from the inhibitor-treated cells were measured as described in the experimental procedure. Data show means ± SDs and are representative of 2 independent experiments. (d) M-Mac were transfected with 100 pmol si-RNA against TAK-1 (si-TAK) or control si-RNA (si-Ctrl) as described in the experimental procedure, and then cultured with or without 100 ng/ml of IL-27 for 48 h. The expression of TAK-1 and p47phox were analyzed by western blotting. (e) si-TAK-1 or si-Ctrl-transfected cells were stimulated with or without 100 ng/ml PMA for 30 min and then superoxide production was monitored as described in the experimental procedure. Data show means ± SDs and are representative of 3 independent experiments. *P < 0.01.

Fig 5: IL-27 enhances phosphorylation of p47phox in macrophages.IL-27 treated and untreated macrophages were stimulated with 100 ng/ml PMA for 20 min at 37 °C and then whole cell lysates were prepared in the presence of a phosphatase inhibitor and proteinase inhibitor. Western blot was performed with anti-phosphorylated p47phox, anti-p47phox, anti-gp91phox, and anti-ß-actin was used for detecting a loading control.