Caspase-1, the Microvascular Endothelium, and Infection

 描述（由申请人提供）：尽管有令人信服的实验证据将炎症与急性呼吸窘迫综合征（ARDS）的发病机制联系起来，但抗炎临床试验系统性地未能证明对患者的有益作用。这种失败通常归因于相互关联的前景，即炎症的泛抑制是有害的，或者抗炎药也抑制保护性应激反应。因此，确定治疗ARDS的新靶点需要了解驱动免疫细胞中促炎反应和非免疫细胞中保护性应激适应程序的关键分子的基本生物学。初步数据显示，除了在巨噬细胞中诱导促炎反应外，Caspase-1蛋白酶激活还保护肺微血管内皮细胞（PMVEC）和肺动脉内皮细胞（PAEC）响应感染的屏障功能。本文提供的其他数据支持一种模型，其中Caspase-1降解PMVEC和PAEC中的糖酵解和线粒体蛋白，作为限制感染诱导的晚期N-糖化终产物（AGEs）和活性氧/氮物质（RS）积累的保护策略。有趣的是，这些研究中使用的模型机会致病菌（铜绿假单胞菌）和嗜血管性病原体（普氏立克次氏体）都部署了抑制Caspase-1激活的同源分泌型磷脂酶A2毒素（ExoU）。所提出的实验将验证以下假设：PMVEC和PAEC中的Caspase-1活化促进糖酵解和线粒体蛋白的降解作为保护屏障功能的适应性应激反应。具体目标1将通过以下方式阐明PMVEC和PAEC中半胱天冬酶-1活化的机制：1.1）使用Split Venus荧光互补报道子在炎性体信号传导水平上定义PMVEC和PAEC感测感染的机制。1.2)使用磷脂酶A2信号传导抑制剂确定ExoU分泌的毒素抑制胱天蛋白酶-1活化的机制。具体目标2将通过以下方式阐明PMVEC和PAEC中Caspase-1诱导的应激反应的机制：2.1）阐明Caspase-1降解的糖酵解和线粒体蛋白，并通过定点突变和酶功能测定验证靶标。2.2)确定Caspase-1是否在感染期间保护PMVEC和PAEC屏障功能。将使用分子和药理学方法下调或上调Caspase-1，然后评估屏障功能、AGEs和RS。糖酵解中间体、AGEs和/或RS的抑制剂将揭示它们在屏障消亡中的作用。具体目标3将通过以下方式将半胱天冬酶-1活化和线粒体功能与ARDS患者结果相关联：3.1）测量免疫细胞和非免疫细胞中的活性半胱天冬酶-1和线粒体呼吸。3.2)患者死亡率和无呼吸机天数与预后的相关性对人类健康的长期影响和转化潜力在于确定目前缺乏的治疗ARDS的疗法的靶点。

 DESCRIPTION (provided by applicant): Despite compelling experimental evidence linking inflammation to the pathogenesis of Acute Respiratory Distress Syndrome (ARDS), anti-inflammatory clinical trials have systematically failed to demonstrate beneficial effects in patients. This failure is often ascribed to the interrelated prospects that pan-suppression of inflammation is deleterious or that anti-inflammatories also inhibit protective stress responses. Thus, identifying novel targets to treat ARDS requires an understanding of the basic biology underlying key molecules that drive both pro-inflammatory responses in immune cells and protective stress adaptation programs in non-immune cells. Preliminary Data show that, in addition to inducing pro-inflammatory responses in macrophages, Caspase-1 protease activation protects Pulmonary Microvascular Endothelial Cell (PMVEC) and Pulmonary Arterial Endothelial Cell (PAEC) barrier function in response to infection. Additional data presented herein support a model in which Caspase-1 degrades glycolytic and mitochondrial proteins in PMVECs and PAECs as a protective strategy that limits accumulation of advanced N-glycation end products (AGEs) and reactive oxygen/nitrogen species (RS) induced by infection. Intriguingly, the model opportunistic pathogen (Pseudomonas aeruginosa) and vasculotropic pathogen (Rickettsia prowazekii) used in these studies both deploy homologous secreted phospholipase A2 toxins (ExoU) that inhibit Caspase-1 activation. The proposed experiments will test the Hypothesis that Caspase-1 activation in PMVECs and PAECs elicits degradation of glycolytic and mitochondrial proteins as an adaptive stress response to protect barrier function. Specific Aim 1 will elucidate mechanisms of Caspase-1 activation in PMVECs and PAECs by: 1.1) Defining the mechanisms by which PMVECs and PAECs sense infection at the level of Inflammasome signaling using a Split Venus fluorescence complementation reporter. 1.2) Determining mechanisms by which the ExoU secreted toxin inhibits Caspase-1 activation using phospholipase A2 signaling inhibitors. Specific Aim 2 will elucidate mechanisms of Caspase-1-induced stress responses in PMVECs and PAECs by: 2.1) Elucidating glycolytic and mitochondrial proteins degraded by Caspase-1 and validating targets by site-directed mutagenesis and enzyme function assays. 2.2) Determining whether Caspase-1 protects PMVEC and PAEC barrier function during infection. Molecular and pharmacologic approaches will be used to either down-regulate or up-regulate Caspase-1 followed by assessment of barrier function, AGEs, and RS. Inhibitors of glycolytic intermediates, AGEs, and/or RS will unveil their roles in barrier demise. Specific Aim 3 will correlate Caspase- 1 activation and mitochondrial function with ARDS patient outcomes by: 3.1) Measuring active Caspase-1 and mitochondrial respiration in immune cells and non-immune cells. 3.2) Correlating outcomes with patient mortality and ventilator-free days. Long-term impact on human health and translational potential lie in identifying targets for therapies to treat ARDS, which are currently lacking.