Oxidative stress enhances the expression of IL-33 in human airway epithelial cells

Abstract

Background: Interleukin-33 (IL-33) is a cytokine belonging to the IL-1 family, and its possible involvement in the pathophysiology of COPD and viral-induced exacerbations has been demonstrated. IL-33 has been shown to be increased in the airway epithelial cells from COPD patients, but the regulating mechanism of IL-33 expression in airway epithelial cells remains largely unknown. In the current study, we examined whether oxidative stress, which participates in the pathogenesis of COPD, affects the expression of IL-33 in airway epithelial cells and also evaluated the effect during viral infection.

Methods: The involvement of oxidative stress in the expression of IL-33, and its signal pathway was examined after stimulation with hydrogen peroxide (H₂O₂), with or without stimulation by polyinosinic-polycytidylic acid [poly (I:C)], a synthetic analogue of dsRNA that mimics viral infection, or rhinovirus infection in NCI-H292 cells and primary human bronchial epithelial cells (HBECs). In addition, the effect of antioxidant, N-acetylcysteine (NAC) in the expression of IL-33 was compared between HBECs from healthy subjects and those from COPD patients.

Results: Treatment with H₂O₂ significantly potentiated IL-33 expression in NCI-H292 cells, and the potentiation was reversed by NAC treatment. Mitogen-activated protein kinase (MAPK) inhibitors, but not nuclear factor-kappa B inhibitors, also significantly decreased the H₂O₂-potentiated IL-33 expression. In addition, H₂O₂ significantly potentiated the poly (I:C)- or rhinovirus-stimulated IL-33 expression. In HBECs from healthy subjects, H₂O₂-potentiated IL-33 expression and its reversal by NAC was also confirmed. Under the condition without H₂O₂-stimulation, treatment with NAC significantly decreased the expression of IL-33 in HBECs from COPD patients, but not in those from healthy subjects.

Conclusions: These results demonstrate that oxidative stress involves in the expression of IL-33 in airway epithelial cells via MAPK signal pathway and it augments IL-33 expression during viral infection. This mechanism may participate in the regulation of IL-33 expression in airway epithelial cells in COPD and the viral-induced exacerbations. Modulation of this pathway could become a therapeutic target for viral-induced exacerbations of COPD.

Keywords: COPD; Exacerbation; IL-33; Oxidative stress; Viral infection.

Fig. 1

Effect of H₂O₂ on the expression of Interleukin-33 (IL-33) in NCI-H292 cells. a-c Effect of H₂O₂ on the gene expression of IL-33 and the cell viability in NCI-H292 cells. a, b Cells were treated with various concentration of H₂O₂. After 4 h incubation, whole cells were harvested and assayed for the gene expression of IL-33 (a) and cell viability (b). c Cells were treated with 200 μM H₂O₂. At various time points after the incubation, whole cells were harvested and assayed. d, e Evaluation of nuclear fraction of IL-33 protein. At various time points after the treatment with 200 μM H₂O₂, the nuclear fraction of cell lysates was obtained. d IL-33 protein in the nuclear fraction was evaluated by immunoblotting. e Each band intensity was assessed by densitometry. Relative intensity was calculated by dividing the IL-33 band intensity by each appropriate lamin A/C band intensity. f Effect of N-acethylcysteine (NAC) on H₂O₂-augmented IL-33 expression. Values are the mean ± SEM (n = 3 - 5). *p < 0.05, ***p < 0.001 compared with the values of vehicle-treated cells

Fig. 2

Involvement of mitogen-activated protein kinase signaling pathways in H₂O₂-potentiated IL-33 expression. a-d Effect of H₂O₂ on the phosphorylation of MAPK (p38, JNK, ERK1/2) in NCI-H292 cells. Cells were treated with NAC or vehicle prior to treatment with H₂O₂. After 15 min, whole cell lysates were obtained. a Phosphorylation of p38, JNK or ERK was evaluated with immunoblotting. b-d Band intensities were assessed by densitometry. Relative intensity was calculated by dividing the phosphorylated p38, JNK or ERK1/2 band intensity by the p38, JNK or ERK1/2 band intensity and the results were indicated as fold change over control. e-g Effect of p38-MAPK inhibitor (SB203580) (e), JNK inhibitor (SP600125) (f) or ERK inhibitor (U0126) (g) on H₂O₂-potentiated IL-33 expression in NCI-H292 cells. Values are the mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the values of vehicle-treated cells

Fig. 3

Effect of H₂O₂ on a synthetic viral dsRNA analogue, poly (I:C)-potentiated IL-33 expression. a NCI-H292 cells were treated with H₂O₂ or vehicle 30 min prior to the treatment with poly (I:C). After 4 h, whole cells were harvested and assayed for the IL-33 gene expression. b Effect of NAC on H₂O₂-augmented IL-33 expression in poly (I:C)-treated cells. Cells were added with NAC before H₂O₂ treatment, and then treated with poly (I:C). Values are the mean ± SEM (n = 3 - 4). ***p < 0.001 compared with the values of vehicle-treated control. ^†††p < 0.001 compared with the values of poly (I:C)-treated control

Fig. 4

Effect of H₂O₂ on viral-potentiated IL-33 expression. Time course (a) or comparison at 4 and 24 h (b) of IL-33 expression after treatment with H₂O₂ and human rhinovirus, RV14 infection. NCI-H292 cells were infected with RV14 in the presence of H₂O₂ or vehicle. At various time points after incubation (a) or after 4 and 24 h (b), whole cells were harvested and assayed for the IL-33 gene expression. c Effect of NAC on H₂O₂-augmented IL-33 expression in RV14-treated cells. Cells were added with NAC before H₂O₂ treatment, and then infected with RV14. After 24 h RV14 infection, whole cells were harvested and assayed. Values are the mean ± SEM (n = 2 - 5). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the values of vehicle-treated cells

Fig. 5

Involvement of oxidative stress in the expression of IL-33 in HBECs from COPD patients. a Effect of H₂O₂-potentiated IL-33 expression and reversal by NAC in human bronchial epithelial cells (HBECs) from healthy subjects. Cells were treated with NAC or vehicle prior to treatment with H₂O₂. After 4 h, whole cells were harvested and assayed for the IL-33 gene expression. b Effect of NAC on the expression of IL-33 in HBECs from healthy subjects and COPD patients. Cells were treated with NAC or vehicle. After 4 h, whole cells were harvested and assayed. The data are expressed as the mean ± SEM of five healthy subjects or six COPD patients. c-e Effect of p38-MAPK inhibitor (SB203580), JNK inhibitor (SP600125) or ERK inhibitor (U0126) on the expression of IL-33 in HBECs from COPD patients. Cells were treated with the MAPK inhibitor or vehicle. After 4 h, whole cells were harvested and assayed. The data is a representative of two independent experiments with four samples performed using HBECs from two COPD patients. **p < 0.01 compared with the values of vehicle-treated cells

Fig. 6

Schematic representation of the effect of oxidative stress on IL-33 expression in airway epithelial cells. H₂O₂ potentiates the expression of IL-33 in human airway epithelial cells. H₂O₂ enhances the phosphorylation of MAPK (p38, JNK, ERK1/2) and a p38 MAPK inhibitor, SB203580, a JNK inhibitor, SP600125, and an ERK1/2 inhibitor, U0126 inhibits the expression of IL-33. Neither an IKK-2 inhibitor, SC-514 nor an IκBα inhibitor, BAY11-7085 affects the H₂O₂-augmented IL-33 expression. H₂O₂ potentiates the expression of IL-33 stimulated by TLR3 ligand, poly (I:C) and rhinoviral-infection, and the potentiation is inhibited by the antioxidant NAC. These data suggest that oxidative stress enhances the expression of IL-33 via MAPK pathway and the expression of IL-33 can be further increased in human airway epithelial cells during virus infections. NAC = N-acetylcysteine. BAY = BAY 11-7085