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

在本PPG中，所有项目和核心都考虑了呼吸衰竭的持续性和 急性呼吸窘迫综合征患者的多器官功能障碍是炎症消退的正常机制失败 和肺修复最近对ARDSnet中登记的患者进行的临床分析支持了这一假设 其中ARDS的“高度炎症”内型与包括死亡在内的不良临床结局相关。的 许多编码炎性细胞因子的基因的表达受转录因子NF-κ B B调节。 因此，了解NF-κ B B活性在肺损伤和修复过程中是如何控制的， 对损伤后肺修复成功或失败的病理生物学的机械见解。 线性泛素组装复合物（LUBAC）是一种E3泛素连接酶，其执行Met-1泛素化 NF-κ B B活化所必需的。在初步的实验中，我们观察到上皮特异性缺失a LUBAC的调节组分（HOIL-1 L）阻止NF-κ B B信号传导并改善小鼠中的结果 甲型流感病毒感染模型。我们提出了额外的初步数据，支持我们的假设， LUBAC促进肺泡上皮细胞和单核细胞源性肺泡上皮细胞中持续的NF-κ B B信号传导 在甲型流感诱导的肺损伤后，巨噬细胞抑制肺修复。 具体目标1。确定抑制LUBAC介导的肺上皮中NF-κ B B活化是否 在从甲型流感感染中恢复的过程中加速肺修复。我们将进行定时实验， 我们将在甲型流感感染恢复期间遗传性抑制LUBAC介导的NF-κ B B信号传导， 使用补充生理和分子技术来测量肺修复。 具体目标2。为了确定LUBAC的激活是否介导了肺中NF-κ B B的激活， 上皮指导单核细胞衍生的巨噬细胞中的修复表型。我们将决定 这些细胞的炎性表型是由变化的微环境（特别是肺）驱动的 上皮，或者这些变化是否是巨噬细胞内的细胞自主性。 具体目标3。为了确定是否增加NF-κ B依赖性炎症基因的表达， 重症肺炎患者的肺泡巨噬细胞和BAL液与30天 mortality.我们将利用成功临床缓解提供的基础设施， 肺炎治疗（CCLT）试验，以确定NF-κ B的表达是否与 肺泡巨噬细胞和BAL液细胞因子中的β B靶基因与临床结局

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.