Endothelial Toll-Like Receptor Signaling and Inflammation

描述（由申请人提供）：脓毒症是感染引起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外，几乎没有开发出其他治疗策略，败血性休克的死亡率仍高达60- 70%。每年约有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细菌性脓毒症的有效治疗方法。已知炎性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，其触发脓毒症病理生理学。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，立即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会聚点，即细胞内GT3 ARF 6发出信号。该会聚点控制细胞-细胞连接蛋白的运输，并且不同于激活免疫应答的典型转录途径（例如，那些激活NF-κ B的）。许多细菌PAMP通过Toll样受体（TLR）信号传导，并且PAMP/TLR信号传导被认为在脓毒症中起关键作用。我们假设，抑制ARF 6将提供一个平台，通过增强血管系统对PAMP/TLR信号传导的弹性来治疗脓毒症，而不会进一步损害免疫系统。我们将通过追求三个目标来检验这一假设。在目标1中，我们将鉴定在PAMP/TLR信号传导中激活ARF 6的上游分子组分。GTP酶的ARF家族被称为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，连接受体与ARF-GEF的衔接蛋白是ARF 6激活所必需的。因此，我们将确定哪些衔接蛋白和ARF-GEFs是PAMP/TLR激活ARF 6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。在目标2中，我们确定PAMP/TLR激活的ARF 6如何增加内皮通透性。我们已经表明，在细胞因子信号传导中，活化的ARF 6通过降低细胞表面的VE-钙粘蛋白水平诱导内皮通透性，从而破坏将内皮细胞保持在一起的粘附连接。在这个目标中，我们将确定是否PAMP/TLR激活的ARF 6同样破坏粘附连接，并将确定直接效应的ARF 6激活。在目标3中，我们将明确确定Arf 6的内皮表达是否是三种不同的细菌性脓毒症小鼠模型中病理性血管渗漏、器官衰竭和死亡所必需的。我们还将确定通过肽或小分子抑制剂阻断ARF 6功能是否可以减少这些脓毒症模型中的血管渗漏、器官衰竭和死亡率。这些目标的成功完成将阐明ARF 6在细菌性脓毒症中的作用，并将决定ARF 6是否是开发治疗细菌性脓毒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.