P6: Th1/Th2 Cytokines promote Nitrite/H2O2-mediated tyrosine nitration in airway epithelial cells: Potential role in severe asthma

Background Protein tyrosine nitration is increased in the airways of asthmatics, particularly in severe disease, where the evidence of a complex adaptive immune process with elements of an active Th1 pathway, in combination with a Th2 signature, is increasingly observed. Although airway 3-nitrotyrosine (3NT) formation is generally attributed to peroxynitrite, formed from NO and superoxide, 3NT can also be generated through NO 2 radical, formed from nitrite and H 2 O 2 . Nitrite is an active metabolite of iNOS-derived NO, while in airway epithelial cells, H2O2 is believed to be primarily produced by Dual oxidase (DUOX)-2. In human airway epithelial cells, Th1 and Th2 cytokines induce iNOS, while DUOX2 and H 2 O 2 are upregulated by Th1 cytokine IFN- γ , and further enhanced by Th2 cytokine IL-13 (Voraphani et al., ATS abstract 2012). Whether the concomitant presence of Th1 and Th2 stimuli could augment nitrative stress is not known. We hypothesized the Th1 cytokine (IFN- γ ), in the face of a Th2 (IL-13) background, would enhance nitrite production through a nitrite/H2O2 pathway, which would contribute to increases in 3NT. Methods Epithelial cells (from bronchial brushings) [1 normal control (NC), 12 mild/moderate (MMA), 6 severe asthmatics (SA)] were cultured in air–liquid interface with IL-13 (1,10 ng/ml) or media for 8 days, with/without IFN- γ (10,100 ng/ml) for the last 72 h. This was followed by 1-h incubation with PBS with/without the following: nitrite (10,25 μM), catalase (H 2 O 2 scavenger, 150 U/ml), or the combination. Nitrite and nitrate levels were measured by the Griess reaction. 3NT levels were quantified in lysates by ELISA. iNOS mRNA and protein were evaluated by RT-PCR and Western blot, respectively. Fresh bronchial epithelial cells from 7 NC, 9 MMA, and 10 SA were also analyzed for 3NT expression. Results Nitrite levels were dose-dependently increased by IFN- γ and IL-13. Although nitrate was the predominant metabolite generated by both cytokines, IFN- γ treatment resulted in a greater ratio of nitrite/nitrate. The presence of IFN- γ , together with low-dose IL-13, synergistically induced iNOS and nitrite expression compared to either cytokine alone. In contrast, the combination did not increase nitrate. Both low-and high-dose IFN- γ increased 3NT levels, while only low-dose IL-13 had an effect. Similar to iNOS and nitrite, 3NT levels were synergistically enhanced in the presence of low-dose IL-13 and IFN- γ . The percent increase in nitrite correlated with the percent increase in 3NT from baseline ( r = 0.7, p γ . Furthermore, 3NT expression increased following nitrite addition, an effect marginally dependent on the presence of IL-13 and IFN- γ , and was attenuated ∼50% with catalase. 3NT levels were elevated in SA epithelial cells ex vivo compared to MMA ( p p Conclusions 3NT expression is increased in SA epithelial cells. In ALI culture, increases in 3NT paralleled increases in iNOS and nitrite, in response to the combination of Th1 and Th2 cytokines. This is similar to our findings on DUOX2 and H 2 O 2 . These results suggest Th1 stimuli, in the presence of a Th2 background, as seen in severe asthma, may play a key role in enhanced nitrative stress through its impact on nitrite/H 2 O 2 -mediated tyrosine nitration. Disclosure Funding: HL69174, AI 67780, Dellenback Fund.