Mechanisms of innate immune response modulation by mechanical ventilation

描述（由申请人提供）：机械通气经常用于支持危重患者，即使在没有明显肺损伤的情况下。然而，最近的一项研究将机械通气暴露与急性呼吸窘迫综合征（ARDS）的后续发展联系起来。机械通气与肺损伤发生的机制尚不清楚。我们已经证明，机械通气可以改变低剂量细菌产物暴露的转录反应，导致过度炎症和肺损伤的早期发展。我们的长期目标是确定机械通气如何调节对微生物病原体的转录反应，作为开发特定干预措施以提高机械通气患者生存率的第一步。这一提议的具体假设是，机械通气激活了激活因子蛋白1 （AP-1）转录因子，从而增加了基因转录，以响应toll样受体（TLRs）对病原体相关分子模式（PAMP）的识别。我们基于以下观察得出这一假设：A)机械通气优先诱导含有AP-1结合序列的启动子区域基因的转录，B)机械通气导致AP-1的核易位，C)机械通气协同增加响应脂多糖（LPS）的基因mRNA水平，脂多糖是革兰氏阴性菌的细胞壁成分。D) c-Jun氨基末端激酶（JNK - AP-1的激酶激活剂）的抑制对其他类型的肺损伤具有保护作用。本提案的具体目的是：1)确定pamp诱导炎症的呼吸机增强是否(a)特定于个体TLR-信号通路和/或(b)依赖于内源性TLR配体的产生。这一目标是通过高度特异性toll样受体激动剂和缺乏特异性TLR信号所必需的MyD88或TRIF适配器蛋白的转基因小鼠的结合来实现的。2)通过(a)测量嵌合小鼠对TLR配体和通气的反应，确定主要对呼吸机诱导的免疫调节反应的肺细胞类型，嵌合小鼠在髓细胞或非髓细胞中缺乏MyD88， (b)测量转基因小鼠对TLR配体和通气的反应，其中远端肺上皮细胞在下游TLR信号通路中存在分离缺陷。3)通过(a)使用染色质免疫沉淀法确定AP-1的哪些蛋白亚基通过机械通气被募集到靶基因启动子，(b)在机械通气和LPS暴露期间抑制AP-1的激活，以及(c)使用RNA干扰确定(a)中确定的特定AP-1亚基的功能，确定AP-1激活在LPS诱导的炎症通气机增强中的作用。与公共卫生的相关性——急性肺损伤（ALI）的发病率为78.9 / 100,000患者年，死亡率为38.5%。基于这些数值，美国每年估计有190,600例ALI病例，其中74,500例相关死亡。此外，ALI的经济影响仅次于ICU护理时间延长和住院总时间以及慢性住院后残疾。这些数据强调了进一步研究的重要性，以帮助了解促进ALI发展的因素。该项目将有助于确定机械通气支持导致肺损伤的机制；从而为未来干预措施的发展提供指导，以降低急性肺损伤的发生率和严重程度。

DESCRIPTION (provided by applicant): Mechanical ventilation is frequently used to support critically ill patients even in the absence of overt lung injury. A recent study, however, has linked exposure to mechanical ventilation with subsequent development of the acute respiratory distress syndrome (ARDS). The mechanism linking mechanical ventilation with initiation of lung injury is unknown. We have shown that mechanical ventilation can alter the transcriptional response to low-dose bacterial product exposure leading to excessive inflammation and early development of lung injury. Our long-term goal is to identify how mechanical ventilation modulates the transcriptional response to microbial pathogens as a first step toward developing specific interventions to improve survival in mechanically ventilated patients. The specific hypothesis for this proposal is that mechanical ventilation activates the activator protein-1 (AP-1) transcription factor, which augments gene transcription in response to pathogen-associated molecular pattern (PAMP) recognition by toll-like receptors (TLRs). We base this hypothesis on the observations that: A) mechanical ventilation preferentially induces transcription of genes with a promoter region containing the AP-1 binding sequence, B) mechanical ventilation causes nuclear translocation of AP-1, C) mechanical ventilation synergistically augments mRNA levels of genes transcribed in response to lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, and D) inhibition of c-Jun amino-terminal kinase (JNK - a kinase activator of AP-1) is protective against other types of lung injury. The specific aims of this proposal are to: 1) Determine whether ventilator augmentation of PAMP-induced inflammation is (a) specific for individual TLR- signaling pathways and/or (b) dependent on generation of endogenous TLR ligands. This aim is accomplished through the combination of highly specific toll-like receptor agonists and genetically modified mice, which lack either the MyD88 or TRIF adapter protein necessary for specific TLR signaling. 2) Determine the lung cell type primarily responsive to ventilator-induced immunomodulation by (a) measuring response to TLR ligands and ventilation in chimeric mice, which lack MyD88 in either myeloid or non- myeloid cells and (b) measuring response to TLR ligands and ventilation in transgenic mice in which distal lung epithelial cells have an isolated defect in downstream TLR signaling pathways. 3) Determine the role of AP-1 activation in ventilator-augmentation of LPS-induced inflammation by (a) using chromatin immunoprecipitation assay to identify which protein subunits of AP-1 are recruited to target gene promoters by mechanical ventilation, (b) inhibiting AP-1 activation during mechanical ventilation and LPS exposure, and (c) using RNA interference to determine the function of specific AP-1 subunits identified in (a). Relevance for public health - The incidence of acute lung injury (ALI) is 78.9 per 100,000 patient-years with a mortality rate of 38.5%. Based on these values, there are an estimated 190,600 cases of ALI in the U.S. each year with 74,500 associated deaths. Additionally, the economic impact of ALI is high secondary to prolonged ICU care and overall length of hospitalization and to chronic post-hospitalization disability. These data highlight the importance of further research to help understand the factors, which promote the development of ALI. This project will help identify mechanisms by which mechanical ventilation support causes lung injury; thereby, providing guidance for the development of future interventions to decrease the incidence and severity of acute lung injury.