An airway epithelial iNOS-DUOX2-thyroid peroxidase metabolome drives Th1/Th2 nitrative stress in human severe asthma

Abstract

Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H(2)O(2). Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H(2)O(2), but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H(2)O(2) formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H(2)O(2) to nitrogen dioxide radical (NO(2)(•)) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS-DUOX2-TPO-NO(2)(•) metabolome drives nitrative stress in HAEC and likely in SA.

Figure 1

Elevated FeNO and epithelial 3NT levels in SA. (a) FeNO levels were increases in SA compared to HC. Overall P<0.01, **P<0.01; Kruskal-Wallis with Bonferroni correction; NS, not significant. Data were shown as median (25–75 percentiles). (b) 3NT levels were highest in fresh bronchial epithelial cells from SA compared to MMA and HC. Overall P<0.001, *P<0.05, ***P<0.001; adjusted for age. Results were shown as mean±SEM.

Figure 2

The combination of low-dose IL-13 and IFN-γ enhances iNOS expression, nitrite, and 3NT levels. ALI-cultured HAEC were treated with low-dose IL-13 (1 ng/ml) or media for 8 d, with/without exposure to IFN-γ (10 ng/ml) for final 72 h. mRNA was harvested for real-time PCR. Total proteins were harvested for Western blot analysis and 3NT measurement. Nitrite levels were measured in the lower supernatants. (a) iNOS mRNA, (b) iNOS protein, (c) nitrite, and (d) 3NT expression were augmented in the presence of IL-13 and IFN-γ compared to either cytokine alone. P for interaction <0.05 for all markers; *P<0.025, **P<0.001; n=16, 15, 16, 19 for iNOS mRNA, iNOS protein, nitrite, and 3NT, respectively. Densitometry values were shown as mean±SEM.

Figure 3

3NT expression changes in parallel with levels of H₂O₂ and nitrite. ALI-cultured HAEC were treated with low-dose IL-13 (1 ng/ml) or media for 8 d, with/without exposure to IFN-γ for the last 72 h. Culture medium from the apical chamber was removed, and 100 µl PBS with/without one of the following was added: catalase (150 U/ml), SOD (150 U/ml), nitrite (10,25 µM), or the combination of catalase and nitrite. After 1-h incubation, extracellular H₂O₂ levels were measured in the upper chamber supernatants. Total proteins were harvested for 3NT measurement. (a) Addition of catalase decreased 3NT levels in the presence/absence of IL-13 and IFN-γ while SOD increased 3NT when cells were stimulated with IL-13 and IFN-γ (10 ng/ml). *P<0.05 vs. no catalase/SOD. (b) Exogenous nitrite upregulated 3NT expression while catalase attenuated this effect. ^#P<0.017 vs. no nitrite, *P<0.05 vs. no catalase. (c) H₂O₂ levels were enhanced in the presence of low-dose IL-13 and IFN-γ (10 ng/ml) compared to either cytokine alone. P for interaction <0.05, *P<0.025, **P<0.001. (d) H₂O₂ production followed a similar trend to 3NT, with the levels decreasing with catalase and increasing with SOD. *P<0.05 vs. no catalase/SOD. All n=6. Data represented mean±SEM.

Figure 4

IFN-γ in combination with low-dose IL-13 enhances DUOX2 expression and knockdown of DUOX2 decreases H₂O₂ as well as 3NT levels. ALI-cultured HAEC were stimulated for 8 d with or without low-dose IL-13 (1 ng/ml) and then stimulated (or not) with IFN-γ (10 ng/ml) for the last 72 h. For knockdown experiment, ALI-cultured cells transfected with DUOX2 siRNA were stimulated with IL-13 (1 ng/ml) and IFN-γ (100 ng/ml) for 48 h. The culture medium from the upper chamber was removed, and 100 µl PBS was added. Extracellular H₂O₂ production was measured in the upper chamber supernatants after 1-h incubation. mRNA was harvested for real-time PCR. Total proteins were harvested for Western blot analysis and 3NT measurement. In the concomitant presence of IL-13 and IFN-γ, there were increases in (a) DUOX2 mRNA and (b) DUOX2 protein over that of either IFN-γ or IL-13 alone. P for interaction< 0.001 for both markers, *P<0.025, **P<0.001; n=16 for DUOX2 mRNA, n=11 for DUOX2 protein. (c) Western blot demonstrating DUOX2 knockdown in the presence of IL-13 and IFN-γ stimulation. Inhibition of DUOX2 by siRNA transfection suppressed (d) H₂O₂, and (e) 3NT production induced by IL-13 and IFN-γ. *P<0.05; n=4. Data represented mean±SEM.

Figure 5

TPO is identified as a highly differentiating gene in epithelial microarrays. Box-whisker plot showing linear-scale microarray gene expression of TPO for HC, MMA, and SA. The boxes represented the mean and standard deviation and the whiskers represented the minimum and maximum values of the distribution. *P<0.05; 2-tailed unpaired t-test vs. HC, **P<0.05; 2-tailed unpaired t-test vs. HC and MMA.

Figure 6

TPO expression is enhanced by IFN-γ. ALI-cultured cells were treated with IL-13 or media for 8 d, with/without exposure to IFN-γ for final 72 h. mRNA was harvested for real-time PCR and total proteins were harvested for Western blot analysis. Both concentrations of IFN-γ (10,100 ng/ml) induced (a) TPO mRNA and (b) protein, while (c) only high-dose IL-13 (10 ng/ml), but not low-dose (1 ng/ml) enhanced TPO mRNA. Overall P<0.001, *P<0.017, **P<0.001. Unlike INOS and DUOX2, IFN-γ (10 ng/ml) in combination with low-dose IL-13 (1 ng/ml) did not further increase (d) TPO mRNA or (e) protein. n=9 for TPO mRNA, n=8 for TPO protein. Results were shown as mean±SEM.

Figure 7

TPO siRNA reduces 3NT formation. ALI-cultured cells transfected with TPO siRNA were stimulated with IL-13 (1 ng/ml) and IFN-γ (100 ng/ml) for 48 h. Total proteins were harvested for Western blot analysis and 3NT measurement. (a) Western blot of TPO knockdown in the presence of IL-13 and IFN-γ stimulation. (b) Suppression of TPO by siRNA transfection reduced 3NT production induced by IL-13 and IFN-γ. *P<0.05; one-tailed t-test, n=3. Data represented mean±SEM.

Figure 8

Enhanced DUOX2 and TPO mRNA in epithelial cells from SA. Expression of (a) DUOX2 mRNA and (b) TPO mRNA were increased in fresh bronchial epithelial cells from SA compared to MMA and HC. Overall P<0.05 for both markers, *P<0.05; adjusted for age. Results were shown as mean±SEM.

Figure 9

Proposed biochemical pathway of 3NT generation in airway epithelia.