Pre-Clinical Evaluation of IRE1beta as a Novel Therapeutic Target for Cystic Fibrosis Airway Mucus Production

囊性纤维化(CF)肺在出生后不久就表现出粘液炎症反应，可能是由病毒引发的 感染和/或吸入。呼吸道合胞病毒(RSV)引起毛细支气管炎，导致呼吸道粘液。 年幼的CF儿童出现梗阻，他们的呼吸道中MUC5B和MUC5AC粘蛋白增加。 由于目前还没有针对CF呼吸道粘液过度产生的治疗方法，因此存在明显的未得到满足的医疗需求 用于针对CF呼吸道粘蛋白合成的治疗。Cf呼吸道上皮炎症触发内质 内质网(ER)应激并激活肌醇需要酶1(IRE1)，该酶以两种亚型α和 β.IRE1α普遍存在，但IRE1β仅在呼吸道和胃肠道的粘液细胞中表达。我们有 结果表明，Ire_1β(而不是Ire_1α)是产生呼吸道粘蛋白所必需的。IRE1是一种内质网跨膜蛋白 有一个管腔结构域(未折叠蛋白质的感受器)和一个带有激酶和核糖核酸酶的细胞质结构域(效应器) 活动。目前尚不清楚ir1的管腔结构域是否感知未折叠的粘蛋白，以及其胞浆是否 结构域介导粘蛋白的产生；然而，我们之前的研究表明粘蛋白的产生触发内质网 应激并激活IRE1β激酶诱导的核糖核酸酶激活。因为激活的ire1核糖核酸酶拼接 X-box结合蛋白-1(XBP-1s)的mRNA，一种上调粘蛋白产生的转录因子，这可能是 为CF呼吸道上皮粘蛋白过度产生提供一种机制。在非粘液细胞中，IRE1激酶被激活 JNK、p38 MAP激酶和NF-B通过蛋白质相互作用，但目前尚不清楚IRE1激酶是否被激活 这些途径与CF呼吸道相关，因为它们可以促进粘蛋白的产生。我们的预赛 数据表明，IRE1β、MUC5AC和MUC5B水平在天然的CF人类呼吸道和在 新鲜分离的人远端呼吸道上皮细胞。野生型IRE1在原代人支气管中的过表达 上皮细胞(HBE)增加粘蛋白的产生，而缺乏激酶和/或的IRE1突变体的过度表达 核糖核酸酶活性降低了粘蛋白的产生。IL-1和肿瘤坏死因子，主要的CF呼吸道细胞因子，不同 影响XBP-1s(仅IL-1增加XBP-1s)和粘蛋白产生(IL-1和GT；肿瘤坏死因子)，提示 它们通过IRE1、β、核糖核酸酶依赖和独立的机制上调粘蛋白的产生。KIRA6，一个IRE1 激酶+核糖核酸酶抑制剂，钝化的IL-1可增加CFHBE中的XBP-1s和粘蛋白。值得注意的是， CFTR调节剂(VX-445、VX-661和VX-770)对粘蛋白的产生没有影响。呼吸道合胞病毒感染HBE 增加XBP-1和粘蛋白的产生/分泌，这些反应被KIRA6钝化。最后，小鼠 感染1型副流感病毒的小鼠出现病毒性毛细支气管炎和呼吸道粘膜阻塞，模拟 呼吸道合胞病毒感染的毛细支气管炎我们的目标是测试IRE1β蛋白结构域在CF气道中的作用 细胞因子-增加CFHBE中粘蛋白的产生。我们还将评估抑制IRE1β的治疗效果 关于呼吸道病毒感染诱导CFHBE和包括一只CF鼠在内的小鼠呼吸道粘蛋白的产生。 我们的研究可能导致IRE1β抑制剂的产生，作为治疗CF气道黏膜阻塞的新药物。

Cystic fibrosis (CF) lungs exhibit mucoinflammatory responses soon after birth, likely triggered by viral infections and/or aspiration. Respiratory syncytial virus (RSV) causes bronchiolitis leading to airway muco- obstruction in young CF children, who exhibit increases in MUC5B and MUC5AC mucins in their airways. Because no treatments are available for CF airway mucus overproduction, there is a clear unmet medical need for therapies that target mucin synthesis in CF airways. CF airway epithelial inflammation triggers endoplasmic reticulum (ER) stress and activates the inositol requiring enzyme 1 (IRE1), which exists in two isoforms, α and β. IRE1α is ubiquitous, but IRE1β is only expressed in mucous cells of the respiratory and GI tracts. We have shown that IRE1β (but not IRE1α) is required for airway mucin production. IRE1 is an ER transmembrane protein with a lumenal domain (sensor of unfolded proteins) and a cytoplasmic domain (effector) with kinase and RNase activities. It is unknown whether the IRE1 lumenal domain senses unfolded mucins and whether its cytoplasmic domains mediate mucin production; however, our previous studies suggested that mucin production triggers ER stress and activates IRE1β kinase-induced RNase activation. Because the activated IRE1 RNase splices the mRNA of X-box binding protein-1 (XBP-1s), a transcription factor that up-regulates mucin production, this may provide a mechanism for CF airway epithelial mucin overproduction. In non-mucous cells, IRE1 kinase activates JNK, p38 MAP kinase and NF-B via protein interactions, but it is unknown whether the IRE1 kinase activates these pathways, which are relevant to CF airways because they can promote mucin production. Our preliminary data indicate that IRE1β, MUC5AC and MUC5B levels are up-regulated in native CF human airways and in freshly isolated CF human distal airway epithelia. Over-expression of wild type IRE1 in primary human bronchial epithelia (HBE) increased mucin production, whereas over-expression of IRE1 mutants that lack kinase and/or RNase activities decreased mucin production. IL-1 and TNF, predominant CF airway cytokines, differently affected XBP-1s (only IL-1 increased XBP-1s) and mucin production (IL-1 > TNF) in HBE, suggesting that they up-regulate mucin production via IRE1β RNase-dependent and independent mechanisms. KIRA6, an IRE1 kinase + RNase inhibitor, blunted IL-1-increased XBP-1s and mucins in CF HBE. Notably, a combination of CFTR modulators (VX-445, VX-661 and VX-770) had no effect on mucin production. RSV infection of HBE increased XBP-1s and mucin production/secretion, and these responses were blunted by KIRA6. Finally, murine parainfluenza virus type 1-infected mice developed viral bronchiolitis and airway muco-obstruction, modeling the bronchiolitis in CF infants infected by RSV. Our aims will test the role of IRE1β protein domains in CF airway cytokine-increased mucin production in CF HBE. We will also evaluate the therapeutic effect of IRE1β inhibition on respiratory virus infection-induced mucin production in CF HBE and murine airways, including a CF mouse. Our studies may lead to the generation of IRE1β inhibitors as novel therapeutics for CF airway muco-obstruction.