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，但抗炎临床试验一直未能系统地证明对患者的有益效果。这种失败通常被归因于相互关联的前景，即泛抑制炎症是有害的，或者消炎药也抑制保护性应激反应。因此，确定治疗ARDS的新靶点需要了解免疫细胞中驱动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背后的基本生物学。初步数据显示，除了在巨噬细胞中诱导促炎反应外，Caspase-1激活还能保护肺微血管内皮细胞(PMVEC)和肺动脉内皮细胞(PAEC)在感染时的屏障功能。本文提供的其他数据支持一个模型，在该模型中，Caspase-1降解PMVECs和PAECs中的糖酵解和线粒体蛋白，作为一种保护策略，限制感染诱导的晚期N-糖基化终产物(AGEs)和活性氧/氮物种(RS)的积累。有趣的是，这些研究中使用的模型条件致病菌(铜绿假单胞菌)和亲血管病原体(普氏立克次体)都部署了同源的分泌型磷脂酶A2毒素(ExoU)来抑制Caspase-1的激活。拟议的实验将检验这一假设，即PMVECs和PAECs中Caspase-1的激活引起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。具体目的1将通过以下几个方面阐明Caspase-1在PMVECs和PAECs中的激活机制：1.1)利用分裂的Venus荧光互补报告，确定PMVECs和PAECs在炎症体信号水平上感知感染的机制。1.2)利用磷脂酶A2信号抑制剂确定ExoU分泌毒素抑制Caspase-1激活的机制。特异性目标2将通过以下方式阐明Caspase-1在PMVECs和PAECs中诱导应激反应的机制：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分析验证靶点。2.2)确定Caspase-1在感染时是否保护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。糖酵解中间产物、AGEs和/或RS的抑制剂将揭示它们在屏障消亡中的作用。特殊目标3将通过以下方式将Caspase-1的激活和线粒体功能与ARDS患者的预后相关联：3.1)测量免疫细胞和非免疫细胞中Caspase-1的活性和线粒体呼吸。3.2)预后与患者死亡率和无呼吸机天数的关系。对人类健康和转化潜力的长期影响在于确定目前缺乏的治疗ARDS的治疗目标。

 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.