Interleukin-27 Enhances the Potential of Reactive Oxygen Species Generation from Monocyte-derived Macrophages and Dendritic cells by Induction of p47phox

Abstract

Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47^phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47^phox upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.

Figure 1. IL-27 treatment enhances NADPH oxidase-2-mediated superoxide production in macrophages.

(a–d) Superoxide Production was monitored by detecting H₂O₂ amounts after stimulation with PMA as described in the Method section. (a) M-Mac were treated with 0 or 100 ng/ml of IL-27 for 24, 48 or 72 h followed by stimulation with PMA, (b) M-Mac were treated with 100 ng/ml of IL-27 for 48 h and then stimulated with PMA for 5, 10, 20, or 30 min at 37 °C. (c,d) M-Mac was cultured with 100 ng/ml IL-27 for 48 h, and then pretreated with apocynin or DPI for 30 min before PMA stimulation (e,f) M-Mac were treated with 100 ng/ml of IL-27 for 48 h. (e) the expression of each subunit mRNA of NADPH oxidase-2 was determined by quantitative RT-PCR. Data shown means ± SE of three independent studies. (f) The expression of p47^phox protein was detected by Western blot. (g) M-Mac were treated with 0 or 100 ng/ml of IL-27 for different durations, or (h) different doses of IL-27 for 48 h. The relative level of expression of p47^phox gene was measured by quantitative RT-PCR. (i,j) M-Mac were treated with CHX for 30 min at 37 °C and then cultured in the presence of 0 or 100 ng/ml of IL-27 for 48 h. The expression of p47^phox protein (i) and mRNA (j) were analyzed by Western blot and quantitative RT-PCR, respectively. (k) M-Mac were incubated with 0 or 100 ng/ml of IL-27 for 48 h and then SOD isotype expression was analyzed by Western blot. (l) M-Mac were treated with 0 or 100 ng/ml of IL-27 for 48 h and then infected with HIV. The HIV infected cells were cultured for 14 days and then HIV replication was monitored by p24 antigen capture kit. Data shown represent means ± SDs of triplicate samples of two independent experiments. *P < 0.01, **P < 0.05 In Western blot analysis, anti-b-actin antibody was used to determine an internal control.

Figure 2. JAK inhibitors suppress the IL-27 induction of p47^phox 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 p47^phox 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-p47^phox antibody. Anti-β 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 p47^phox 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.

Figure 3. IL-27 increases the expression of p47^phox in macrophages.

(a) M-Mac were transfected with either control si-RNA (si-Ctlr) or si-RNA targeting p47^phox (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-p47^phox or anti-β-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 p47^phox expression vector for 6 h, and then the expression levels of p47^phox were determined by Western blot. (d) H₂O₂ induction by PMA stimulation was detected by ROS assays following transfection of either the empty or p47^phox 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 p47^phox 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.

Figure 4. IL-27 enhances phosphorylation of p47^phox 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 p47^phox, anti-p47^phox, anti-gp91^phox, and anti-β-actin was used for detecting a loading control.

Figure 5. The expression of p47^phox is increased in IL-27-induced macrophages (I-Mac).

Monocytes were differentiated into macrophages in the absence (M-Mac) or presence of IL-27 (I-Mac). After differentiation, cells were incubated with or without IL-27. (a) M-mac, I-Mac, untreated M-Mac and IL-27-treated M-Mac were stimulated with or without 100 ng/ml PMA for 30 min at 37 °C and then ROS activity was measured by detection of secreted H₂O₂. Data shown represent means ± SD of triplicate samples. (b) The expression levels of p47^phox were determined by western blotting for M-Mac, I-Mac, untreated M-Mac and IL-27-treated M-Mac. (c) M-Mac and I-Mac were stimulated with or without PMA for 30 min, and then cell lysates were subjected to 2D gel electrophoresis. Expression of p47^phox was detected by anti-p47^phox and anti-phosphorylated S304 p47^phox antibodies. (d) Whole cell lysates of M-Mac and I-Mac were prepared using RIPA buffer. Western blot analyses were performed using anti-SOD1, SOD2, SOD3 or anti-β-actin antibodies. The intensity of the band was analyzed by NIH Image J and normalized each SOD intensity with β-Actin.

Figure 6. 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 p47^phox and gp91^phox 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 p47^phox, gp91^phox, and β-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.

Figure 7. A schematic model of IL-27 effect on superoxide generation.

IL-27 binds to IL-27 receptor composing of gp130 and WSX1. The binding induces JAK/STAT activation followed by enhancement of p47^phox expression in macrophages. PMA stimulation activates PKC and augments phosphorylation of p47^phox, subsequently the highly phosphorylated p47^phox is involved in increase in the superoxide generation.