Mechanism of action for the epithelial-specific ER stress sensor IRE1β in regulating intestinal homeostasis and host defense

项目摘要 我们的首要目标是了解上皮特异性ER应激传感器IRE 1b（ERN 2）及其 普遍表达的副作用IRE 1a（ERN 1）有助于肠道内稳态和宿主防御。 ER对衬于粘膜表面，特别是在肠道中的上皮细胞独特的显著适应是： ER应力传感器IRE 1b的表达，IRE 1b是主要的和进化上最保守的ER的近缘 应力传感器IRE 1a。然而，我们最近发现，IRE 1b在酶促反应中的功能与IRE 1a不同。 活性，使其能够作为ER应激反应中IRE 1a信号传导的非竞争性抑制剂。我们的新 正在进行的研究表明，在体内，IRE 1b在杯状细胞分化和组装中起关键作用， 这两种活动都依赖于正常的肠道微生物群：肠道微生物及其代谢产物。 产品刺激IRE 1b的表达，IRE 1b反过来影响组装、分类和功能输出 肠道微生物的繁殖。 这些数据构成了我们的中心假设，即IRE 1b作为环境诱导的先天免疫调节因子， 上皮宿主防御通过IRE 1a影响ER应激反应，然后杯状细胞发育， 粘液组装和宿主上皮-微生物相互作用，这些作用形成结肠的结构和功能。 在目标1中，我们将使用重组蛋白以及生物化学和生物物理方法来测试IRE 1a和 IRE 1b可能被肠腔因子激活，解释IRE 1b如何与IRE 1a寡聚化，并阐明 结构特征和翻译后修饰修饰了这种装配、IRE 1b信号传导和机制 用于IRE 1b的激活和IRE 1a功能的抑制。 在目标2中，我们将使用野生型和IRE 1b-/-小鼠和从这些动物制备的类结肠来确定IRE 1b如何在小鼠体内表达。 将肠道微生物群与上皮应激和发育反应联系起来，并确定核心微生物途径 以及调节IRE 1b表达和功能的代谢物。 在目标3中，我们将解释IRE 1b的缺乏如何减弱急性感染C的恢复。啮齿动物。我们 发现IRE 1b-/-小鼠的结肠粘液层组装受损，并存在生态失调模拟缺陷 在患有溃疡性结肠炎（UC）的人类中观察到的结肠粘膜中。正如啮齿类柠檬酸杆菌所模拟的那样， IRE 1b-/-小鼠粘液层的改变与正常清除后的恢复受损有关。 病原体我们将（i）测试是否是肠道微生物群失调，或未解决的上皮ER应激，或两者兼而有之， 持续炎症后C. IRE 1b-/-小鼠的啮齿类感染;和（ii）确定是否获得了微生物群 从患有UC表型的人中，这些效应并不能诱导IRE 1b、杯状细胞发育或有效的免疫抑制剂。 当在无菌wt IRE 1b +/+小鼠中重构时，粘液屏障。 内质网应激与慢性炎症和其他肠道疾病有关。这些研究的结果 将提供宿主-微生物群相互作用的信息，这些相互作用调节上皮发育和宿主防御。

PROJECT SUMMARY Our overarching goal is to understand how the epithelial-specific ER stress sensor IRE1b (ERN2) and its ubiquitously expressed paralogue IRE1a (ERN1) contribute to intestinal homeostasis and host defense. A notable adaptation of the ER unique to epithelial cells lining mucosal surfaces, especially in the gut, is expression of the ER stress sensor IRE1b, a close paralogue of the major and most evolutionarily conserved ER stress sensor IRE1a. We recently discovered, however, that IRE1b functions distinctly from IRE1a in enzymatic activity, enabling it to act as a non-competitive inhibitor of IRE1a signaling in the ER stress response. Our new and ongoing studies demonstrate in vivo that IRE1b operates critically in goblet cell differentiation and assembly of the mucus barrier - with both activities dependent on the normal gut microbiota: gut microbes and their products stimulate IRE1b expression, and IRE1b in turn affects the assembly, taxonomy, and functional output of colonizing gut microbes. These data frame our central hypothesis that IRE1b acts as an environmentally induced mediator of innate epithelial host defense by affecting the ER stress response via IRE1a, and then goblet cell development, mucus assembly, and host epithelial-microbe interactions that shape the structure and function of the colon. In Aim 1, we will use recombinant proteins and biochemical and biophysical approaches to test how IRE1a and IRE1b may be activated by gut lumenal factors, explain how IRE1b oligomerizes with IRE1a, and elucidate what structural features and post-translational modifications modify this assembly, IRE1b signaling, and mechanisms for IRE1b activation and suppression of IRE1a function. In Aim 2, we will use wt and IRE1b-/- mice and colonoids prepared from these animals to determine how IRE1b links the gut microbiota with epithelial stress and development responses and identify core microbial pathways and metabolites that regulate IRE1b expression and perhaps function. In Aim 3, we will explain how the lack of IRE1b attenuates recovery from acute infection with C. rodentium. We found that IRE1b-/- mice have impaired assembly of the colonic mucus layer and dysbiosis - mimicking defects in the colonic mucosa seen in humans with ulcerative colitis (UC). As modeled by Citrobacter rodentium, the altered mucus layer in IRE1b-/- mice was associated with impaired recovery following normal clearance of the pathogen. We will (i) test if the dysbiotic gut microbiota, or unresolved epithelial ER stress, or both, drive the sustained inflammation following C. rodentium infection in IRE1b-/- mice; and (ii) determine if microbiota obtained from humans with UC phenocopy these effects and fail to induce IRE1b, goblet cell development, or an effective mucus barrier when reconstituted in germ-free wt IRE1b+/+ mice. ER stress is implicated in the chronic inflammatory and other intestinal diseases. The outcomes of these studies will be informative of the host-microbiota interactions that regulate epithelial development and host defense.