Dual Oxidase in Airway Epithelial Injury and Inflammation

项目总结 NADPH氧化酶DUOX1在呼吸道上皮细胞中显著表达，并参与先天性 通过产生活性氧(ROS)对伤害或其他环境触发物的反应机制 激活氧化还原依赖的信号通路。在这个项目的当前资金周期中，我们确定了 DUOX1作为天然上皮细胞对变应原反应的关键介质，通过促进上皮细胞分泌 Alarmin IL-33和随后的2型炎症的激活。我们还观察到增强的上皮DUOX1 在过敏性哮喘患者中，以及DUOX1在房屋各种关键特征发展中的关键作用 尘螨(HDM)诱导的小鼠过敏性呼吸道炎症，如黏液化生，上皮下纤维化， 和呼吸道高反应性。DUOX1的这些作用在很大程度上与氧化还原依赖的激活有关 Src家族激酶和表皮生长因子受体(EGFR)信号转导，这两个都是在 过敏性炎症和气道重塑，部分是通过在这些激酶中介导半胱氨酸氧化而实现的。在.期间 在这些研究中，我们注意到DUOX1不仅在呼吸道上皮内起作用，而且在非呼吸道上皮中也存在。 上皮细胞类型，如肺泡巨噬细胞，并似乎参与巨噬细胞极化和 IL-33介导的促纤维化介质，如IL-13和转化生长因子-β，参与了气道重塑。第一 这一更新申请的目的是描绘DUOX1在HDM的各个方面的细胞特异性作用- 诱导变态反应性炎症和重塑，特别是DUOX1在替代中的潜在作用 中性粒细胞(N_2)极化或巨噬细胞(M_2)激活。目标2是确定DUOX1的氧化还原敏感靶点 并对DUOX1依赖的半胱氨酸氧化调控酶的分子机制进行了表征 Src的功能，使用分子动力学模拟和各种半胱氨酸变体的实验研究。 最后，基于之前发现的硫醇反应性亲电体可以抑制DUOX1的激活，目标3将是 开发基于半胱氨酸的多肽靶向方法，从药物上抑制DUOX1的活性，从而填补 对DUOX1选择性抑制剂的需求未得到满足，这些抑制剂可能用于临床开发治疗重症 哮喘。

PROJECT SUMMARY The NADPH oxidase DUOX1 is prominently expressed within the respiratory epithelium and contributes to innate response mechanisms to injury or other environmental triggers, by generating reactive oxygen species (ROS) and activating redox-dependent signaling pathways. In the present funding cycle of this project, we identified DUOX1 as a critical mediator of innate epithelial responses to allergens, by promoting epithelial secretion of the alarmin IL-33 and subsequent activation of type 2 inflammation. We also observed enhanced epithelial DUOX1 in subjects with allergic asthma, and a critical role for DUOX1 in development of various critical features of house dust mite (HDM)-induced allergic airway inflammation in mice, such as mucus metaplasia, subepithelial fibrosis, and airway hyperresponsiveness. These actions of DUOX1 were largely related to redox-dependent activation of Src family kinases and epidermal growth factor receptor (EGFR) signaling, both well-recognized factors in allergic inflammation and airway remodeling, in part by mediating cysteine oxidation within these kinases. During these studies, we noted that DUOX1 is not only operative within the airway epithelium, but is also present in non- epithelial cell types such as alveolar macrophages, and appears to be involved in macrophage polarization and IL-33-mediated pro-fibrotic mediators such as IL-13 and TGF-β that are involved in airway remodeling. The first aim of this renewal application is to delineate the cell-specific actions of DUOX1 on various aspects of HDM- induced allergic inflammation and remodeling, specifically focusing on a potential role for DUOX1 in alternative neutrophil (N2) polarization or macrophage (M2) activation. Aim 2 is to identify redox-sensitive targets of DUOX1 and characterize the molecular mechanisms by DUOX1-dependent cysteine oxidation regulates the enzymatic function of Src, using molecular dynamics simulations and experimental studies with various cysteine variants. Finally, based on previous findings that thiol-reactive electrophiles can inhibit DUOX1 activation, aim 3 will be to develop peptide-based cysteine-targeted approaches to pharmacologically inhibit DUOX1 activity, thus filling an unmet need for DUOX1-selective inhibitors that may be used for clinical development in treatment of severe asthma.