Project 3: Targeting linear ubiquitination to attenuate inflammation and promote repair after viral pneumonia

在这个PPG中，所有的项目和核心都考虑到呼吸衰竭的持续性和 ARDS患者的多器官功能障碍是正常炎症消退机制的失败 以及肺部修复。这一假设在临床上得到了最近一项对ARDSNet患者的分析的支持 ARDS的“炎症性”内型与包括死亡在内的不良临床结局相关。这个 许多编码炎性细胞因子的基因的表达受转录因子NF-B的调节。 因此，了解在肺损伤和修复过程中如何控制核因子-B的活性可能会提供 对损伤后肺修复成功或失败的病理生物学的机械性见解。 线性泛素组装复合体(LUBAC)是一种执行Met-1泛素化的E3泛素连接酶 是激活核因子-B所必需的。在初步实验中，我们观察到上皮特异性缺失 LUBAC的调节成分(HOIL-1L)可抑制NF-B信号转导并改善小鼠的预后 甲型流感病毒感染模型。我们提供了额外的初步数据来支持我们的假设 LUBAC促进肺泡上皮和单核细胞源性肺泡持续存在的NF-B信号转导 巨噬细胞抑制甲型流感肺损伤后的肺修复。 具体目的1.确定抑制LUBAC介导的肺上皮细胞NF-B活化是否 在甲型流感康复期间，感染会加速肺部修复。我们将在中国进行计时实验 在甲型流感恢复期间，我们将从基因上抑制LUBAC介导的NF-B信号，并 使用互补的生理学和分子技术来测量肺修复。 特异性目的2.确定LUBAC的激活是否介导了肺组织中NF-B的激活 上皮引导单核细胞来源的巨噬细胞的修复表型。我们将确定是否 这些细胞的炎症表型是由不断变化的微环境驱动的--特别是肺 这些变化是否在巨噬细胞内是细胞自主的。 特定目的3.确定核因子-B依赖的炎症基因表达增加是否 重症肺炎患者肺泡巨噬细胞和肺泡灌洗液与30天的相关性 死亡率。我们将利用成功的临床响应所提供的基础设施 肺炎治疗(SCRIPT)试验，以确定NF-1的表达是否与 肺泡巨噬细胞和肺泡灌洗液细胞因子中B靶基因与临床结局的关系。

In this PPG, all of the Projects and Cores consider the persistence of respiratory failure and the development of multiple organ dysfunction in patients with ARDS as a failure of normal mechanisms of inflammation resolution and lung repair. This hypothesis is clinically supported by a recent analysis of patients enrolled in the ARDSnet where a “hyerinflammatory” endotype of ARDS was associated with poor clinical outcomes including death. The expression of many genes encoding inflammatory cytokines is regulated by the transcription factor NF-B. Hence, understanding how NF-B activity is controlled over the course of lung injury and repair is likely to provide mechanistic insights into the pathobiology of successful or failed lung repair after injury. The Linear Ubiquitin Assembly Complex (LUBAC) is an E3 ubiquitin ligase that performs Met-1 ubiquitination necessary for NF-B activation. In preliminary experiments, we observed that epithelial specific deletion of a modulatory component of LUBAC (HOIL-1L) prevents NF-B signaling and improves outcomes in a murine model of influenza A virus infection. We present additional preliminary data that support our hypothesis that LUBAC promotes persistent NF-B signaling in the alveolar epithelium and monocyte-derived alveolar macrophages to inhibit lung repair after influenza A induced lung injury. Specific Aim 1. To determine whether inhibiting LUBAC-mediated NF-B activation in the lung epithelium during recovery from influenza A infection accelerates lung repair. We will perform timed experiments in which we will genetically inhibit LUBAC mediated NF-B signaling during recovery from influenza A infection and use complementary physiologic and molecular techniques to measure lung repair. Specific Aim 2. To determine whether activation of LUBAC mediated NF-B activation in the lung epithelium directs reparative phenotypes in monocyte-derived macrophages. We will determine whether the inflammatory phenotype of these cells is driven by the changing microenvironment—specifically the lung epithelium, or whether these changes are cell autonomous within the macrophage. Specific Aim 3. To determine whether increased expression of NF-B-dependent inflammatory genes in alveolar macrophages and BAL fluid from patients with severe pneumonia is associated with 30 day mortality. We will take advantage of the infrastructure provided by the Successful Clinical Response in Pneumonia Therapy (SCRIPT) trial to determine whether there is an association between the expression of NF- B target genes in alveolar macrophages and BAL fluid cytokines with clinical outcomes.