Mechanisms of innate immune response modulation by mechanical ventilation

描述（由申请人提供）：即使在没有明显肺损伤的情况下，机械通气也经常用于支持危重患者。然而，最近的一项研究表明，暴露于机械通气与随后发生的急性呼吸窘迫综合征（ARDS）有关。机械通气与肺损伤发生的机制尚不清楚。我们已经表明，机械通气可以改变对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。该建议的具体假设是，机械通气激活激活蛋白-1（AP-1）转录因子，其响应于toll样受体（TLR）对病原体相关分子模式（PAMP）的识别而增强基因转录。我们的假设基于以下观察结果：A）机械通气优先诱导具有包含AP-1结合序列的启动子区的基因的转录，B）机械通气引起AP-1的核转位，C）机械通气协同地增加响应于脂多糖（LPS）（革兰氏阴性细菌的细胞壁组分）而转录的基因的mRNA水平，和D）抑制c-Jun氨基末端激酶（JNK -AP-1的激酶激活剂）对其它类型的肺损伤具有保护作用。该提议的具体目的是：1）确定PAMP诱导的炎症的呼吸机增强是否（a）对个体TLR信号传导途径具有特异性和/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的toll样受体激动剂和遗传修饰的小鼠的组合来实现的，所述小鼠缺乏特异性TLR信号传导所必需的MyD 88或TRIF衔接蛋白。2)通过（a）测量嵌合小鼠中对TLR配体和通气的响应，和（B）测量转基因小鼠中对TLR配体和通气的响应，所述嵌合小鼠在骨髓或非骨髓细胞中缺乏MyD 88，和所述转基因小鼠中远端肺上皮细胞在下游TLR信号传导途径中具有孤立缺陷，来确定主要响应于呼吸机诱导的免疫调节的肺细胞类型。3)通过（a）使用染色质免疫沉淀测定来鉴定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活化，和（c）使用RNA干扰来确定（a）中鉴定的特定AP-1亚基的功能，来确定AP-1活化在LPS诱导的炎症的呼吸机增强中的作用。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%。根据这些数据，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的经济影响是继发于长期ICU护理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.