Tiotropium Bromide Has a More Potent Effect Than Corticosteroid in the Acute Neutrophilic Asthma Mouse Model

Abstract

Background: Neutrophilic asthma (NeuA) is usually resistant to corticosteroids. Tiotropium bromide (TIO) is a bronchodilator that is used as an add-on therapy to inhaled corticosteroid and long-acting β2 agonist in asthma treatment. However, the role of TIO in NeuA is not fully known. Thus, the aim of this study was to evaluate the effect of TIO on NeuA compared to that of corticosteroids.

Methods: C57BL/6 female mice were sensitized with ovalbumin and lipopolysaccharide to induce neutrophilic inflammation. Dexamethasone (DEX) was administered on days 14, 17, 20, and 23. TIO was inhaled on days 21, 21, and 23. On day 24, mice were sacrificed. Airway hyper-responsiveness, levels of cytokines in bronchoalveolar lavage (BAL) and lung homogenates, and lung tissue histopathology were compared between the two groups.

Results: Neutrophil counts, T helper 2 cells (TH2)/TH17 cytokines, and pro-inflammatory cytokine in BAL fluids were elevated in the NeuA group. TIO group showed lower total cells, neutrophil counts, and eosinophil counts in BAL fluids than the DEX group (p<0.001, p<0.05, and p<0.001, respectively). Airway resistance was attenuated in the TIO group but elevated in the NeuA group (p<0.001). Total protein, interleukin (IL)-5, and IL-17A levels in BAL fluids were lower in the TIO group than in the NeuA group (all p<0.05).

Conclusion: TIO showed more potent effects than DEX in improving airway inflammation and attenuating airway resistance in NeuA.

Keywords: Corticosteroid; Neutrophilic Asthma; Tiotropium Bromide.

Fig. 1.

Schematic flow of acute neutrophilic asthma mouse model study. Mice were sensitized with ovalbumin (OVA) on days 0 and 7. OVA was challenged on days 14, 15, 21, 22, and 23 intranasally. Neutrophilic inflammation was induced by lipopolysaccharides (LPS) which was administered on days 18, 21, and 23 intranasally. Dexamethasone was used for challenge on days 14, 17, 20, and 23 intraperitoneally. Tiotropium bromide was inhaled on days 21, 22, and 23.

Fig. 2.

Airway resistance of acute neutrophilic asthma according to medication. Airway resistance is determined by forced oscillation technique. It was measured in response to an increasing dose of methacholine. Tests were proceeded in four groups (control, O+L, O+L+D, and O+L+T). The level of airway resistance was elevated in the O+L group. However, the O+L+D group did not show improvement of airway hyperresponsiveness. The O+L+T group showed significant attenuated airway resistance compared to the O+L group and the O+L+D group. O+L vs. all: **p <0.01, ***p<0.001; O+L+D vs. O+L+T: ^#p<0.05, ^###p<0.001. O: ovalbumin; L: lipopolysaccharides; D: dexamethasone; T: Tiotropium bromide.

Fig. 3.

Lung inflammation status of neutrophilic asthma according to the drug used for challenged, such as dexamethasone or Tiotropium bromide. (A, B) Compared to the O+L group, the dexamethasone group (O+L+D) did not show significant improvement in the level of total cells, neutrophils, eosinophils, or total protein in bronchoalveolar lavage (BAL) fluid. On the contrary, the tiotropium bromide group (O+L+T) showed significant improvement of total cells including neutrophils in BAL fluid and total protein level in BAL fluid. O+L vs. all: *p<0.05, **p <0.01, ***p<0.001; O+L+D vs. O+L+T: ^###p<0.001. (C, D) Inflammation score calculated by hematoxylin and eosin staining of lung tissue histopathology showed improvement both in the O+L+D group and the O+L+T group. O: ovalbumin; L: lipopolysaccharides; D: dexamethasone; T: Tiotropium bromide.

Fig. 4.

Inflammatory cytokines related to neutrophilic inflammation such as interleukin (IL)-5 and IL-17A in bronchoalveolar lavage (BAL) fluid were attenuated by Tiotropium bromide (A, C). Levels of IL-5 and IL-17A were not significantly attenuated by dexamethasone (O+L+D). After dexamethasone and Tiotropium bromide were applied, levels of IL-22 and IL-4 were not differ from those in the neutrophilic asthma group (O+L) (B, D). O+L vs. all: *p<0.05, **p<0.01. O: ovalbumin; L: lipopolysaccharides; D: dexamethasone; T: Tiotropium bromide.

Fig. 5.

Inflammatory cytokines in lung tissue homogenates showed a declining tendency after treatment with Tiotropium bromide (TIO) without having a clinical significance. T helper cytokines such as interleukin (IL)-1β and IL-6 from lung homogenates (A, B) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ (C, D) showed a tendency of decline but no clinical significance after treatment with TIO compared to the neutrophilic asthma group. O+L vs. all: *p<0.05, ***p <0.001. O: ovalbumin; L: lipopolysaccharides; D: dexamethasone; T: Tiotropium bromide.