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. 2013 Dec:65:38-46.
doi: 10.1016/j.freeradbiomed.2013.06.012. Epub 2013 Jun 11.

Dual oxidase regulates neutrophil recruitment in allergic airways

Sandra Chang  1 , Angela Linderholm  1 , Lisa Franzi  1 , Nicholas Kenyon  1 , Helmut Grasberger  2 , Richart Harper  3
Affiliations

Dual oxidase regulates neutrophil recruitment in allergic airways

Sandra Chang et al. Free Radic Biol Med. 2013 Dec.

Abstract

Enhanced reactive oxygen species production in allergic airways is well described and correlates with increased airway contractions, inflammatory cell infiltration, goblet cell metaplasia, and mucus hypersecretion. There is also an abundance of interleukin-4/interleukin-13 (IL-4/IL-13)- or interleukin-5-secreting cells that are thought to be central to the pathogenesis of allergic asthma. We postulated that the dual oxidases (DUOX1 and DUOX2), members of the nicotinamide adenine dinucleotide phosphate oxidase family that release hydrogen peroxide (H2O2) in the respiratory tract, are critical proteins in the pathogenesis of allergic airways. DUOX activity is regulated by cytokines, including IL-4 and IL-13, and DUOX-mediated H2O2 influences several important features of allergic asthma: mucin production, IL-8 secretion, and wound healing. The objective of this study was to establish the contribution of DUOXs to the development of allergic asthma in a murine model. To accomplish this goal, we utilized a DUOXA-deficient mouse model (Duoxa(-/-)) that lacked maturation factors for both DUOX1 and DUOX2. Our results are the first to demonstrate evidence of DUOX protein and DUOX functional activity in murine airway epithelium. We also demonstrate that DUOXA maturation factors are required for airway-specific H2O2 production and localization of DUOX to cilia of fully differentiated airway epithelial cells. We compared wild-type and Duoxa(-/-) mice in an ovalbumin exposure model to determine the role of DUOX in allergic asthma. In comparison to DUOX-intact mice, Duoxa(-/-) mice had reduced mucous cell metaplasia and lower levels of TH2 cytokine levels in bronchoalveolar fluid. In addition, increased airway resistance in response to methacholine was observed in Duoxa(+/+) mice, as expected, but was absent in Duoxa(-/-) mice. Surprisingly, Duoxa(-/-) mice had decreased influx of neutrophils in bronchoalveolar fluid and lung tissue sections associated with a lower level of the chemotactic cytokine IL-6. These findings suggest that DUOX-derived H2O2 has an important role in signaling neutrophils into allergic airways.

Keywords: Asthma; BALF; DPI; DUOX; Dual oxidase; Free radicals; IL; L-T(4); Lung; Murine; Neutrophil; ROS; T(H); T-helper cell; TLR; Toll-like receptor; bronchoalveolar lavage fluid; diphenyleneiodonium; dual oxidase; interleukin; levothyroxine; reactive oxygen species.

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Figures

Figure 1
Figure 1
DUOX expression and activity in isolated mouse airway cells. (A) Western blot analysis confirmed DUOX expression in isolated epithelial cells from Duoxa+/+ and Duoxa−/−mice. (B) Duoxa+/+ and Duoxa−/− mouse airway cells were isolated from tracheas and immediately analyzed for DPI-inhibitable H2O2 production by Amplex® Red analysis. (C) Immunohistochemistry on airway epithelial cells using a mouse anti-DUOX antibody in ovalbumin treated animals (arrowheads indicate cilia).
Figure 2
Figure 2
DUOX is necessary for increased airway hyperresponsiveness. Percent change in compliance (A) or resistance (B) was determined in Duoxa+/+ (+/+) and Duoxa−/− (−/−) mice exposed to two weeks of either filtered air or ovalbumin. Percent change values were calculated by dividing the remainder of compliance or resistance determined at the highest dose of methacholine ( 2mg/mL) minus compliance or resistance determined after exposure to aerosolized saline (baseline value) by the baseline value times 100. Data are shown as mean±SEM from six mice in each group; * = p< 0.05.
Figure 3
Figure 3
DUOX induces mucin expression in mouse lung epithelium after allergen challenge. (A) Periodic acid-Schiff (PAS) staining of paraffin-embedded lung sections obtained from Duoxa+/+ (+/+) and Duoxa−/− (−/−) male mice after two weeks of aerosolized ovalbumin exposure in sensitized animals. Similar airway levels are shown to ensure equivalent representation of hyperplastic potential between groups (200x) (B) Quantification of PAS-positive staining was determined by counting the number of PAS-positive epithelial cells normalized to total epithelial cells for Duoxa−/− (-/-) and Duoxa+/+ (+/+) mice. Five fields incorporating the upper airway were randomly chosen for counting, *=p<0.05.
Figure 4
Figure 4
DUOX affects neutrophilic, but not eosinophilic, airway inflammation after allergen challenge. (A) Leukocytes were collected from the airway compartment by bronchioalveolar lavage (BAL) and the number of live cells was determined by trypan blue exclusion. The number of live cells was compared between ovalbumin-exposed (Ova) versus filtered air-exposed (Air) Duoxa−/− (-/-) and Duoxa+/+ (+/+) mice. (B) Leukocytes were collected from the airway compartment by bronchoalveolar lavage after two-weeks of aerosolized ovalbumin exposure and cell differentials were determined visually based on cell morphology. The percent of each cell type was compared between ovalbumin-exposed Duoxa−/− (-/-) and Duoxa+/+ (+/+) mice. (C) Absolute neutrophil counts in ovalbumin-exposed Duoxa−/− (-/-) and Duoxa+/+ (+/+) mice was calculated by multiplying neutrophil percentage with total cell number from BAL. Data are shown as mean±SEM from six mice in each group; * = p< 0.05 using two-way ANOVA and Bonferroni post-test correction.
Figure 5
Figure 5
Duoxa−/− mice had lower neutrophil influx into multiple compartments of airway tissue. Paraffin-embedded lung sections were obtained from ovalbumin-sensitized Duoxa+/+ (+/+) and Duoxa−/− (-/-) male mice after two weeks of aerosolized ovalbumin exposure. Lung sections were stained with hematoxylin and eosin (HE) for morphometric analysis of neutrophilic influx. The presence of neutrophils was verified by immunohistochemical staining using with rat anti-Ly6 antibody (αLy6). Representative images (400X) of the alveolar (A), peribronchiolar (B), and perivascular (C) compartments are shown. Black arrows highlight positive neutrophil staining.
Figure 5
Figure 5
Duoxa−/− mice had lower neutrophil influx into multiple compartments of airway tissue. Paraffin-embedded lung sections were obtained from ovalbumin-sensitized Duoxa+/+ (+/+) and Duoxa−/− (-/-) male mice after two weeks of aerosolized ovalbumin exposure. Lung sections were stained with hematoxylin and eosin (HE) for morphometric analysis of neutrophilic influx. The presence of neutrophils was verified by immunohistochemical staining using with rat anti-Ly6 antibody (αLy6). Representative images (400X) of the alveolar (A), peribronchiolar (B), and perivascular (C) compartments are shown. Black arrows highlight positive neutrophil staining.
Figure 5
Figure 5
Duoxa−/− mice had lower neutrophil influx into multiple compartments of airway tissue. Paraffin-embedded lung sections were obtained from ovalbumin-sensitized Duoxa+/+ (+/+) and Duoxa−/− (-/-) male mice after two weeks of aerosolized ovalbumin exposure. Lung sections were stained with hematoxylin and eosin (HE) for morphometric analysis of neutrophilic influx. The presence of neutrophils was verified by immunohistochemical staining using with rat anti-Ly6 antibody (αLy6). Representative images (400X) of the alveolar (A), peribronchiolar (B), and perivascular (C) compartments are shown. Black arrows highlight positive neutrophil staining.
Figure 6
Figure 6
Neutrophilic inflammation was similarly attenuated in all lung compartments from Duoxa−/− mice. Paraffin-embedded lung sections were obtained from ovalbumin-sensitized Duoxa+/+ (+/+) and Duoxa−/− (-/-) male mice after two weeks of aerosolized ovalbumin exposure. Lung sections were stained with hematoxylin and eosin (HE) to determine the percentage of neutrophils in each of four lung compartments (see Methods). Leukocyte populations in 10 high-powered fields for each compartment were counted and neutrophil percentage was determined by dividing the total neutrophils counted by the total number of leukocytes. Data are shown as mean±SEM for the perivascular (PV), vascular (V), peribronchiolar (PB), and alveolar (A) compartments, * = p=< 0.05.
Figure 7
Figure 7
DUOX does not impact expression of IL-8 homologues after ovalbumin exposure. BALF was obtained from ovalbumin-sensitized Duoxa+/+ (+/+) and Duoxa−/− (−/−) mice immediately after two weeks of aerosolized ovalbumin exposure. ELISA was used to determine levels of MIP-2 and KC in BALF supernatant. Data represent mean±SEM from six animals in each group.
Figure 8
Figure 8
TH2 cytokine levels are reduced in Duoxa−/− mice. Multiplex analyses were used to determine cytokine levels in BALF supernatant from ovalbumin exposed Duoxa+/+ (+/+) and Duoxa−/− (−/−) mice. For each cytokine, cytokine levels from Duoxa−/− mice are shown as a percent of wildtype levels. Data represent mean±SEM from six animals in each group. Mean cytokine levels in pg/mL from each group are shown in parentheses. * = p<0.05.
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