Leukotriene B4 receptors mediate the production of IL‐17, thus contributing to neutrophil‐dominant asthmatic airway inflammation

To the editor, Asthma phenotypes can be classified according to the inflammatory immune cell type that infiltrates the airways, such as eosinophilic, neutrophilic, or mixed eosinophilic/neutrophilic inflammation.1 Generally, eosinophilic inflammation mostly occurs in mild‐to‐mod‐ erate asthma, while neutrophilic inflammation is seen in more se‐ vere asthma phenotypes.1 Steroids, the cornerstone medication for asthma, were reported to increase neutrophil‐dominant airway inflammation by interrupting neutrophil apoptosis and enhancing neutrophil activation.1 Thus, in some asthmatic patients, an increase in neutrophil number in the sputum is associated with persistent asthma and acute asthma exacerbations2; this condition is called steroid‐resistant severe asthma. Recently, IL‐17 was shown to be as‐ sociated with severe neutrophilic asthma development and exacer‐ bation, and IL‐17 levels in serum and sputum were shown to increase with disease severity.3,4 Meanwhile, a previous study demonstrated that leukotriene B4 (LTB4) is present at higher concentrations in spu‐ tum of patients with severe asthma, who have relatively pronounced neutrophil influx, than in those with mild asthma.5 Furthermore, LTB4 receptors, BLT1 and BLT2, were suggested to contribute to the development of asthmatic airway inflammation.6,7 In the present study, we examined whether BLT1/2‐cascade plays a role in the de‐ velopment of neutrophil‐dominant airway inflammation. Details for materials and methods are provided in this article's online supporting information (Data S1). To generate a neutrophil‐dominant pulmonary inflammation model, mice were immunized with 1 or 10 μg of LPS and 75 μg of OVA on days 0, 1, 2, and 7 and then challenged with 50 μg of OVA on days 14, 15, 21, and 22. Mice were killed on day 24 (Figure S1).8 Lung inflammation increased upon immunization with LPS/OVA compared to controls, as observed in the histology analysis (Figure S1). Then, we tested the efficacy of steroid (dexamethasone) in this model. Airway inflammation, mucus secretion, and the recruitment of total immune cells, neutrophils, eosinophils, and lymphocytes were significantly suppressed by dexamethasone in OVA‐induced mice (Figure S1) but were only partially reduced by dexamethasone in LPS/OVA‐induced mice (Figure S1). Thus, these results suggested that our LPS/OVA‐induced airway inflammation model is suitable for studying neutrophil‐dominant allergic pulmonary inflammation


Leukotriene B 4 receptors mediate the production of IL-17, thus contributing to neutrophil-dominant asthmatic airway inflammation
To the editor, Asthma phenotypes can be classified according to the inflammatory immune cell type that infiltrates the airways, such as eosinophilic, neutrophilic, or mixed eosinophilic/neutrophilic inflammation. 1 Generally, eosinophilic inflammation mostly occurs in mild-to-moderate asthma, while neutrophilic inflammation is seen in more severe asthma phenotypes. 1 Steroids, the cornerstone medication for asthma, were reported to increase neutrophil-dominant airway inflammation by interrupting neutrophil apoptosis and enhancing neutrophil activation. 1 Thus, in some asthmatic patients, an increase in neutrophil number in the sputum is associated with persistent asthma and acute asthma exacerbations 2 ; this condition is called steroid-resistant severe asthma. Recently, IL-17 was shown to be associated with severe neutrophilic asthma development and exacerbation, and IL-17 levels in serum and sputum were shown to increase with disease severity. 3,4 Meanwhile, a previous study demonstrated that leukotriene B4 (LTB 4 ) is present at higher concentrations in sputum of patients with severe asthma, who have relatively pronounced neutrophil influx, than in those with mild asthma. 5 Furthermore, LTB 4 receptors, BLT1 and BLT2, were suggested to contribute to the development of asthmatic airway inflammation. 6,7 In the present study, we examined whether BLT1/2-cascade plays a role in the development of neutrophil-dominant airway inflammation.
Details for materials and methods are provided in this article's online supporting information (Data S1).
To generate a neutrophil-dominant pulmonary inflammation model, mice were immunized with 1 or 10 μg of LPS and 75 μg of OVA on days 0, 1, 2, and 7 and then challenged with 50 μg of OVA on days 14, 15, 21, and 22. Mice were killed on day 24 ( Figure S1). 8 Lung inflammation increased upon immunization with LPS/OVA compared to controls, as observed in the histology analysis ( Figure   S1). Then, we tested the efficacy of steroid (dexamethasone) in this model. Airway inflammation, mucus secretion, and the recruitment of total immune cells, neutrophils, eosinophils, and lymphocytes were significantly suppressed by dexamethasone in OVA-induced mice ( Figure S1) but were only partially reduced by dexamethasone in LPS/OVA-induced mice ( Figure S1). Thus, these results suggested that our LPS/OVA-induced airway inflammation model is suitable for studying neutrophil-dominant allergic pulmonary inflammation F I G U R E 1 The LTB 4 receptors BLT1 and BLT2 are critical for neutrophil-dominant airway inflammation and IL-17 production. A, RNA was isolated from lungs, and BLT1 and BLT2 transcript levels were assessed by RT-PCR. B, The fixed lungs were stained with H&E and PAS. Peribronchial and perivascular lung inflammation was measured and scored. C, Immune cells in BALF were obtained using cytospin and stained with H&E. D, The levels of IL-17 in BALF were analyzed using ELISA. All quantitative data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 that is less sensitive to dexamethasone ( Figure S2). Previously, IL-17 was shown to be closely associated with the pathogenesis of severe neutrophilic asthmatic inflammation, and IL-17 levels in serum and sputum increase with disease severity. 3 In agreement with these previous reports, IL-17 levels were significantly increased in BALF from mice with LPS/OVA-induced pulmonary inflammation ( Figure   S3). The levels of IL-4, IL-5, and IL-13 were not increased in BALF of LPS/OVA-induced mice ( Figure S4). To examine the contribution of IL-17, an anti-IL-17 neutralizing antibody (100 μg/mouse) was intraperitoneally administered 1 hour before each challenge. Airway inflammation, mucus secretion, and the infiltration of immune cells were markedly reduced by the anti-IL-17 neutralizing antibody ( Figures S2, S3). Together, these results suggested that IL-17 contributes to neutrophil-dominant airway inflammation and inflammatory immune cell recruitment. LTB 4 levels are higher in exhaled breath condensates of severe asthma patients than in those of mild asthma patients. 5 Therefore, we examined the role of the LTB 4 receptors BLT1 and BLT2 in IL-17 production in the neutrophil-dominant pulmonary inflammation model. We observed increased expression of BLT1 and BLT2 in the lungs of LPS/OVA-induced mice ( Figure 1A). To this aim, the BLT1 inhibitor U75302 (10 μg/mouse) or the BLT2 inhibitor LY255283 (10 mg/kg) was intraperitoneally injected 1 hour before every challenge. Airway inflammation was markedly reduced by U75302 or LY255283, as determined by histological analysis and quantitative analysis of inflammation scores ( Figure 1B). Additionally, mucus secretion and the accumulation of total immune cells, neutrophils, eosinophils, and lymphocytes in BALF were highly enhanced by LPS/ OVA administration, and this effect was suppressed by U75302 or LY255283 ( Figure 1B,C). In addition, the increased IL-17 level was suppressed by treatment with the BLT1 or BLT2 inhibitor ( Figure 1D).
Together, these results suggested that BLT1 and BLT2 contribute to IL-17 production and airway inflammation in neutrophil-dominant pulmonary inflammation. LTB 4 (Figure 2A,B). Histology analysis showed that MK886 or baicalein reduced airway inflammation compared to F I G U R E 2 Upregulation of BLT2 ligands, LTB 4 and 12(S)-HETE, is associated with neutrophil-dominant airway inflammation and IL-17 production. A, Protein was isolated from lungs, and 5-/12-lipoxygenase was assessed by Western blotting. B, LTB 4 and 12(S)-HETE levels in serum were analyzed using ELISAs. C, The fixed lungs were stained with H&E and PAS. Peribronchial and perivascular lung inflammation was measured and scored. D, Immune cells in BALF were obtained using cytospin and stained with H&E. E, The levels of IL-17 in BALF were analyzed using ELISA. All quantitative data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 [Colorfigure can be viewed at wileyonlinelibrary.com] control ( Figure 2C). Additionally, the administration of MK886 or baicalein significantly reduced the infiltration of total immune cells, neutrophils, eosinophils, lymphocytes, macrophages, and the levels of IL-17 as well as mucus secretion ( Figure 2C

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The authors declare that they have no conflicts of interest.