Data Sheet 1_Mast cells are essential in the development of exposure-associated exercise-induced bronchoconstriction in a mouse model.docx

BackgroundCold air and air pollution are known triggers to induce symptoms in exercise-induced bronchoconstriction (EIB). Mast cells are hypothesized to be a key player in the pathogenesis of EIB. This study aims to investigate the role of mast cells using mast cell deficient (Cpa3cre/+) mice and with mast cell stabilizers (Doxantrazole) in an exposure-associated mouse model of EIB.
MethodsMale Cpa3cre/+ mice and wild type littermates or BALB/c mice were exposed to a submaximal running protocol in cold environment (4°C) or resting period (room temperature) 5 days for 3 weeks after oropharyngeal challenge with saline or 0.1 mg/ml diesel exhaust particles (DEP). BALB/c mice were intraperitoneally injected with 16.5 mg/kg Doxantrazole or placebo (0.5% NaHCO3) during the last week. Twenty-four hours after the last running or resting session, lung function, lung inflammation and immune mediated response was determined.
ResultsInhibition of mast cells by Doxantrazole or mice lacking functional mast cells (Cpa3cre/+), resulted in blunting of bronchial hyperreactivity, both in acute breathing pattern and in hyperreactivity to increasing doses of methacholine. Neutrophilic inflammation was still present after Doxantrazole treatment, but not in Cpa3cre/+ mice. These results were similar in neutrophil extracellular traps and neutrophil-linked cytokines and chemokines. Macrophage activaty was also reduced in the absence of functional mast cells.
ConclusionMast cells are crucial in the development of bronchial hyperreactivity and macrophage activation. Additionally, they have a complex interplay with neutrophilic inflammation. These findings highlight the potential of mast cell modulation as therapeutic strategy in exposure-associated EIB.

背景：已知冷空气与空气污染可诱发运动诱发性支气管收缩（exercise-induced bronchoconstriction, EIB）患者出现症状。学界推测肥大细胞（mast cells）是运动诱发性支气管收缩发病机制中的关键参与者。本研究旨在利用肥大细胞缺陷型（Cpa3cre/+）小鼠及肥大细胞稳定剂（Doxantrazole），在暴露相关的运动诱发性支气管收缩小鼠模型中探究肥大细胞的作用。
方法：选取雄性肥大细胞缺陷型（Cpa3cre/+）小鼠、同窝野生型小鼠或BALB/c小鼠，经口咽部给予生理盐水或0.1 mg/ml柴油颗粒物（diesel exhaust particles, DEP），随后连续3周，每日于寒冷环境（4℃）或静息状态（室温）下进行亚极量跑台运动方案。BALB/c小鼠于实验最后一周，腹腔注射16.5 mg/kg Doxantrazole或安慰剂（0.5%碳酸氢钠溶液）。末次运动或静息24小时后，检测小鼠肺功能、肺部炎症及免疫介导的应答反应。
结果：通过Doxantrazole抑制肥大细胞，或使用功能缺陷型肥大细胞的Cpa3cre/+小鼠，均可减弱支气管高反应性，包括急性呼吸模式改变以及对递增剂量乙酰甲胆碱（methacholine）的高反应性。经Doxantrazole处理后，肺部仍存在中性粒细胞性炎症，但Cpa3cre/+小鼠中未出现该现象。中性粒细胞胞外陷阱（neutrophil extracellular traps）及中性粒细胞相关细胞因子、趋化因子的检测结果与上述发现一致。在缺乏功能性肥大细胞的情况下，巨噬细胞活性同样降低。
结论：肥大细胞在支气管高反应性及巨噬细胞活化的发生发展中发挥关键作用。此外，肥大细胞与中性粒细胞性炎症存在复杂的相互作用。本研究结果表明，肥大细胞调控有望成为暴露相关运动诱发性支气管收缩的治疗策略。