Endothelial Toll-Like Receptor Signaling and Inflammation

描述（由申请人提供）：败血症是对感染的灾难性全身炎症反应。尽管进行了大量的研究，但除了非特异性支持治疗之外，很少有治疗策略被开发出来，脓毒性休克病例的死亡率仍然高达60- 70%。每年大约有75万美国人感染败血症，其中90%以上的病例是由于细菌感染引起的炎症、血管渗漏、水肿、器官衰竭和死亡。我们的长期挑战是找到治疗细菌性败血症的有效方法。众所周知，炎症细胞因子和病原体相关分子模式（PAMPs）诱导血管不稳定和水肿，从而引发脓毒性病理生理。我们的初步数据表明，各种细胞因子和PAMPs对血管屏障的直接、即时和破坏性作用是由同源受体介导的，这些受体通过一个共同的汇聚点——细胞内GTPase ARF6发出信号。这个汇聚点控制着细胞-细胞连接蛋白的运输，与激活免疫反应的典型转录途径（例如，激活NF-kappaB的途径）不同。许多细菌PAMPs通过toll样受体（TLR）发出信号，而PAMP/TLR信号被认为在败血症中起着至关重要的作用。我们假设抑制ARF6将通过增强血管系统对PAMP/TLR信号的恢复能力而不进一步损害免疫系统，为治疗败血症提供一个平台。我们将通过追求三个目标来检验这一假设。在Aim 1中，我们将确定在PAMP/TLR信号传导中激活ARF6的上游分子成分。ARF家族的gtpase被称为ARF- gef的鸟嘌呤核苷酸交换因子（gef）激活，我们已经证明在细胞因子激活过程中，连接受体和ARF- gef的适配器蛋白是ARF6激活所必需的。因此，我们将确定哪些适配蛋白和arf - gef是ARF6的PAMP/TLR激活所必需的，并确定这些蛋白是否是诱导内皮通透性所必需的。在Aim 2中，我们确定了PAMP/ tlr激活的ARF6如何增加内皮通透性。我们已经证明，在细胞因子信号传导中，激活的ARF6通过降低细胞表面ve -钙粘蛋白水平诱导内皮通透性，从而破坏将内皮细胞连接在一起的粘附连接。在这个目的中，我们将确定PAMP/TLR激活的ARF6是否同样会破坏粘附连接，并将确定ARF6激活的直接效应物。在Aim 3中，我们将明确确定在三种不同的细菌性脓毒症小鼠模型中，Arf6的内皮表达是否为病理性血管泄漏、器官衰竭和死亡所必需。我们还将确定通过肽或小分子抑制剂阻断ARF6功能是否可以减少这些脓毒症模型中的血管泄漏、器官衰竭和死亡率。这些目标的成功完成将阐明ARF6在细菌性败血症中的作用，并将决定ARF6是否是开发治疗细菌性败血症药物的有希望的靶点。

DESCRIPTION (provided by applicant): Sepsis is a catastrophic systemic inflammatory response to infection. Despite intense study, few therapeutic strategies other than nonspecific supportive care have been developed and death rates remain as high as 60- 70% in cases of septic shock. Approximately 750,000 Americans contract sepsis each year and more than 90% of these cases are due to bacterial infections that trigger inflammation, vascular leak, edema, organ failure, and death. Our long-term challenge is to find an effective therapy for bacterial sepsis. It is known that inflammatory cytokines and pathogen-associated molecular patterns (PAMPs) induce the vascular instability and edema that trigger septic pathophysiology. Our preliminary data suggest that the direct, immediate, and disruptive effects of various cytokines and PAMPs on the vascular barrier are mediated by cognate receptors that signal via a common convergence point, the intracellular GTPase ARF6. This convergence point controls trafficking of cell-cell junction proteins and is distinct from the canonical transcriptional pathwys that activate the immune response (e.g., those activating NF-kappaB). Many bacterial PAMPs signal through toll-like receptors (TLRs), and PAMP/TLR signaling is thought to play a crucial role in sepsis. We hypothesize that inhibiting ARF6 will offer a platform for treating sepsis by enhancing the resilience of the vascular system to PAMP/TLR signaling without further compromising the immune system. We will test this hypothesis by pursuing three aims. In Aim 1, we will identify the upstream molecular components that activate ARF6 in PAMP/TLR signaling. The ARF family of GTPases is activated by guanine nucleotide exchange factors (GEFs) known as ARF-GEFs, and we have shown that during cytokine activation, adapter proteins that link the receptor to the ARF-GEF are required for ARF6 activation. Therefore, we will identify which adapter proteins and ARF-GEFs are required for PAMP/TLR activation of ARF6 and determine whether these proteins are required for the induction of endothelial permeability. In Aim 2, we determine how PAMP/TLR-activated ARF6 functions to increase endothelial permeability. We have shown that in cytokine signaling, activated ARF6 induces endothelial permeability by reducing VE-cadherin levels at the cell surface, thus disrupting the adherens junctions that hold endothelial cells together. In this aim, we will determine whether PAMP/TLR activation of ARF6 likewise disrupts adherens junctions and will identify the direct effectors of ARF6 activation. In Aim 3, we will definitively determine whether the endothelial expression of Arf6 is required for pathologic vascular leak, organ failure, and death in three different mouse models of bacterial sepsis. We will also determine whether blocking ARF6 function by peptide or small molecule inhibitors can reduce vascular leak, organ failure, and mortality rates in these models of sepsis. The successful completion of these aims will elucidate the role ARF6 plays in bacterial sepsis and will dictate whether ARF6 is a promising target for developing drugs that can treat bacterial sepsis.