Caspase-1, the Microvascular Endothelium, and Infection

Caspase-1、微血管内皮和感染

• 批准号: 9043174
• 负责人: JONATHON PETER AUDIA
• 金额: $ 37.88万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9043174
• 关键词: Adult Respiratory Distress Syndrome; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Biological Assay; Biology; CASP1 gene; Case Study; Cell Death; Cell physiology; Cells; Cellular Stress; Clinical Trials; Cysteine; Data; Endothelial Cells; Endothelium; Enzymes; Eukaryotic Cell; Failure; Fluorescence; Generations; Glycolysis; Glycolysis Inhibition; Goals; Health; Homologous Gene; Human; Immune; Immunoblotting; Infection; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Interleukin-18; Life; Link; Lung; Measures; Mediating; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mutagenesis; Nitrogen; Outcome; Oxygen; Pathogenesis; Patient-Focused Outcomes; Patients; Pattern recognition receptor; Peptide Hydrolases; Phospholipase A2; Positioning Attribute; Prevalence; Process; Pseudomonas aeruginosa; Pulmonary Edema; Recovery; Reporter; Resolution; Respiration; Rickettsia prowazekii; Role; Severities; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Stem cells; Stress; Testing; Toxin; Ventilator; Venus; base; biological adaptation to stress; combat; cytokine; extracellular; glycation; inhibitor/antagonist; interleukin-1beta-converting enzyme inhibitor; macrophage; monocyte; mortality; novel; outcome prediction; pathogen; programs; receptor; research study; respiratory; response; scaffold; targeted treatment; therapeutic development

 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.

 描述（由适用提供）：尽管令人信服的实验证据将感染与急性呼吸窘迫综合征（ARDS）的发病机理联系起来，但抗炎临床试验仍未证明对患者的有益作用。这种失败通常被称为相互关联的前景，即炎症的泛抑制是有害的，或者抗炎也抑制了受保护的应激反应。这是确定治疗ARDS的新靶标需要了解驱动免疫细胞中促炎反应的基本生物学基本生物学，并保护非免疫细胞中的应激适应程序。初步数据表明，除了诱导巨噬细胞促炎反应外，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）屏障响应感染。本文提供的其他数据支持了一个模型，在该模型中，caspase-1在PMVEC和PAEC中降解糖酵解和线粒体蛋白是一种保护性策略，该策略限制了受感染引起的高级N-糖化终极产物（AGES）和反应性氧/氮种（RS）的积累。有趣的是，这些研究中使用的模型机会病原体（铜绿假单胞菌）和血管纤维素病原体（Rickettsia prowazekii）均部署抑制caspase-1激活的同源性磷脂酶A2毒素（Exou）。提出的实验将检验以下假设：PMVEC和PAECS中的caspase-1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。特定的目标1将通过以下方式阐明PMVEC和PAECS中caspase-1激活的机制：1.1）定义PMVEC和PAECS使用分裂的金星荧光完成报告剂在炎症体信号水平上感染的机制。 1.2）确定外来分泌毒素使用磷脂酶A2信号抑制剂抑制caspase-1激活的机制。具体目标2将阐明caspase-1诱导的PMVEC和PAECS中的应激反应的机制：2.1）阐明caspase-1降解的糖酵解和线粒体蛋白，并通过位置定向的诱变和酶函数函数验证靶标。 2.2）确定caspase-1在感染过程中是否保护PMVEC和PAEC屏障功能。分子和药物方法将用于下调或上调caspase-1，然后评估屏障功能，年龄和/或RS，将公布其在屏障灭亡中的作用。特定的目标3将通过以下方式将caspase-1激活和线粒体功能与ARDS患者结局相关联：3.1）在免疫细胞和非免疫细胞中测量活性caspase-1和线粒体呼吸。 3.2）将结局与患者死亡率和无呼吸机相关联。长期对人类健康和翻译潜力的影响在于确定目前缺乏治疗ARD的疗法的靶标。