Abstract：
BACKGROUND: The muscarinic M3 receptor antagonist, tiotropium, has a bronchodilatory effect on asthma patients. Additionally, tiotropium inhibits allergic airway inflammation and remodeling in a murine asthma model. However, the underlying mechanisms of this M3 receptor antagonist remain unclear. Therefore, we investigated the effect of muscarinic M3 receptor blockage on M2 macrophage development during allergic airway inflammation.
METHODS: BALB/c mice were sensitized and challenged with ovalbumin to develop a murine model of allergic airway inflammation mimicking human atopic asthma. During the challenge phase, mice were treated with or without tiotropium. Lung cells were isolated 24 h after the last treatment and gated using CD68-positive cells. Relm-α and Arginase-1 (Arg1) (M2 macrophage markers) expression was determined by flow cytometry. Mouse bone marrow mononuclear cell-derived macrophages (mBMMacs) and human peripheral blood mononuclear cells (PBMCs)-derived macrophages were stimulated with IL-4 and treated with a muscarinic M3 receptor antagonist in vitro.
RESULTS: The total cells, eosinophils, and IL-5 and IL-13 levels in BAL fluids were markedly decreased in the asthma group treated with tiotropium compared to that in the untreated asthma group. The Relm-α and Arg1 expression in macrophages was reduced considerably in the asthma group treated with tiotropium compared to that in the untreated asthma group, suggesting that the development of M2 macrophages was inhibited by muscarinic M3 receptor blockage. Additionally, muscarinic M3 receptor blockage in vitro significantly inhibited M2 macrophage development in both mBMMacs- and PBMCs-derived macrophages.
CONCLUSIONS: Muscarinic M3 receptor blockage inhibits M2 macrophage development and prevents allergic airway inflammation. Moreover, muscarinic M3 receptors might be involved in the differentiation of immature macrophages into M2 macrophages.
METHODS: BALB/c mice were sensitized and challenged with ovalbumin to develop a murine model of allergic airway inflammation mimicking human atopic asthma. During the challenge phase, mice were treated with or without tiotropium. Lung cells were isolated 24 h after the last treatment and gated using CD68-positive cells. Relm-α and Arginase-1 (Arg1) (M2 macrophage markers) expression was determined by flow cytometry. Mouse bone marrow mononuclear cell-derived macrophages (mBMMacs) and human peripheral blood mononuclear cells (PBMCs)-derived macrophages were stimulated with IL-4 and treated with a muscarinic M3 receptor antagonist in vitro.
RESULTS: The total cells, eosinophils, and IL-5 and IL-13 levels in BAL fluids were markedly decreased in the asthma group treated with tiotropium compared to that in the untreated asthma group. The Relm-α and Arg1 expression in macrophages was reduced considerably in the asthma group treated with tiotropium compared to that in the untreated asthma group, suggesting that the development of M2 macrophages was inhibited by muscarinic M3 receptor blockage. Additionally, muscarinic M3 receptor blockage in vitro significantly inhibited M2 macrophage development in both mBMMacs- and PBMCs-derived macrophages.
CONCLUSIONS: Muscarinic M3 receptor blockage inhibits M2 macrophage development and prevents allergic airway inflammation. Moreover, muscarinic M3 receptors might be involved in the differentiation of immature macrophages into M2 macrophages.
摘要：
背景：毒蕈碱M3受体拮抗剂，噻托溴铵,对哮喘患者有支气管扩张作用。此外,噻托溴铵在小鼠哮喘模型中抑制过敏性气道炎症和重塑。然而,该M3受体拮抗剂的潜在机制尚不清楚.因此,我们研究了毒蕈碱M3受体阻断对过敏性气道炎症中M2巨噬细胞发育的影响.
方法：BALB/c小鼠致敏并用卵清蛋白攻击，以建立模仿人类特应性哮喘的过敏性气道炎症小鼠模型。在挑战阶段,用或不用噻托溴铵治疗小鼠。在最后一次治疗后24小时分离肺细胞并使用CD68阳性细胞门控。通过流式细胞术测定Relm-α和精氨酸酶-1(Arg1)(M2巨噬细胞标记物)的表达。用IL-4刺激小鼠骨髓单核细胞衍生的巨噬细胞（mBMMacs）和人外周血单核细胞（PBMC）衍生的巨噬细胞，并在体外用毒蕈碱M3受体拮抗剂处理。
结果：总细胞，嗜酸性粒细胞,与未治疗的哮喘组相比,噻托溴铵治疗的哮喘组BAL液中IL-5和IL-13水平显著降低.与未经治疗的哮喘组相比，噻托溴铵治疗的哮喘组巨噬细胞中的Relm-α和Arg1表达显著降低，提示M2巨噬细胞的发育受到毒蕈碱M3受体阻断的抑制。此外,毒蕈碱M3受体阻断体外显着抑制mBMMacs和PBMC衍生的巨噬细胞中M2巨噬细胞的发育。
结论：毒蕈碱M3受体阻断抑制M2巨噬细胞发育并预防过敏性气道炎症。此外，毒蕈碱M3受体可能参与未成熟巨噬细胞向M2巨噬细胞的分化。
方法：BALB/c小鼠致敏并用卵清蛋白攻击，以建立模仿人类特应性哮喘的过敏性气道炎症小鼠模型。在挑战阶段,用或不用噻托溴铵治疗小鼠。在最后一次治疗后24小时分离肺细胞并使用CD68阳性细胞门控。通过流式细胞术测定Relm-α和精氨酸酶-1(Arg1)(M2巨噬细胞标记物)的表达。用IL-4刺激小鼠骨髓单核细胞衍生的巨噬细胞（mBMMacs）和人外周血单核细胞（PBMC）衍生的巨噬细胞，并在体外用毒蕈碱M3受体拮抗剂处理。
结果：总细胞，嗜酸性粒细胞,与未治疗的哮喘组相比,噻托溴铵治疗的哮喘组BAL液中IL-5和IL-13水平显著降低.与未经治疗的哮喘组相比，噻托溴铵治疗的哮喘组巨噬细胞中的Relm-α和Arg1表达显著降低，提示M2巨噬细胞的发育受到毒蕈碱M3受体阻断的抑制。此外,毒蕈碱M3受体阻断体外显着抑制mBMMacs和PBMC衍生的巨噬细胞中M2巨噬细胞的发育。
结论：毒蕈碱M3受体阻断抑制M2巨噬细胞发育并预防过敏性气道炎症。此外，毒蕈碱M3受体可能参与未成熟巨噬细胞向M2巨噬细胞的分化。
