Extracellular vesicle-driven neutrophilic inflammation in cystic fibrosis lungs

项目摘要/摘要 中性粒细胞是人类最丰富的白细胞亚群。目前针对中性粒细胞的治疗方法是 仅限于阻止它们在组织中招募的药物，使它们的功能基本未受影响。我们的长期合作 目标是发展关于人类中性粒细胞生物学和功能的新知识，并从中创新 治疗以中性粒细胞引起的顽固性炎症为主的疾病，包括囊性纤维化(CF)。 Cf是欧洲人后裔中最常见的常染色体疾病，由cftr突变引起。 上皮沟。最近的研究表明，CFTR功能障碍会导致局部粘液淤积阻塞， 紧随其后的是中性粒细胞招募，矛盾的是，机会性感染。这一悖论源于 事实是，大多数中性粒细胞在体内招募到CF气道经历过度活跃的颗粒释放，并成为 免疫调节和代谢许可，但未能杀死微生物，在功能上的命运，我们称之为“严峻”。 使用人体呼吸道炎症的器官型模型，我们先前展示了幼稚的血液中性粒细胞 在体外跨上皮细胞迁移到CF患者的气道液中时，可使患者获得严峻的命运。 这里提出的关于严峻适应的进一步初步研究支持了上游的必要作用 细胞外小泡(EV)存在于携带长非编码RNA Malat-1的气道液中，并且 组蛋白脱乙酰基酶11的转录激活和线粒体重写的下游含义 (HDAC11)和cMyc作为关键调节因子。值得注意的是，我们发现严酷的中性粒细胞在体外产生 分泌二次EV，进而在新一波中性粒细胞中诱导严峻的适应。此外，我们还展示了 抗Malat-1和HDAC11的siRNA及小分子药物SIS17对HDAC11的抑制作用 使严重的中性粒细胞的颗粒释放和杀菌以及它们对次级EV的释放正常化。 我们的目标是全面描述慢性阻塞性肺疾病中性粒细胞严酷适应的机制。我们的中央 假设慢性慢性支气管炎是一个前馈过程，在这个过程中 中性粒细胞释放EV，导致新招募的中性粒细胞面临严峻的命运。 这项研究介绍了一种新的创新的中性粒细胞驱动的人肺炎症的基本范式 电动汽车、Malat-1、HDAC11和cMyc的关键作用，以及解构这一范式(模型)的创新方法 用于大量生产肺招募的中性粒细胞，优化的工具来表征肺EVS，以及药物-/RNA/ 以中性粒细胞为靶点的siRNA干预)。我们预计这项研究将在我们的基础上产生革命性的发现 了解并有能力治疗慢性中性粒细胞炎症和慢性化脓性肺炎及其他疾病。

PROJECT SUMMARY / ABSTRACT Neutrophils are the most abundant leukocyte subset in humans. Current therapies targeting neutrophils are limited to drugs that block their recruitment to tissues, leaving their functions mostly untouched. Our long-term goal is to develop new knowledge on neutrophil biology and function in humans and derive from it innovative treatments for conditions dominated by intractable neutrophil-driven inflammation, including cystic fibrosis (CF). CF is the most frequent autosomal disease in people of European descent, caused by mutations in the CFTR epithelial channel. Recent research has shown that CFTR dysfunction causes local mucus stasis obstruction, followed by neutrophil recruitment, and paradoxically, opportunistic infections. This paradox stems from the fact that most neutrophils recruited to CF airways in vivo undergo hyperactive granule release, and become immunomodulatory and metabolically licensed, yet fail to kill microbes, in a functional fate we dubbed “GRIM”. Using an organotypic model of human airway inflammation, we showed previously that naïve blood neutrophils can be made to acquire the GRIM fate upon transepithelial migration in vitro into airway fluid from CF patients. Further preliminary studies of GRIM adaptation presented here support an obligate upstream role for extracellular vesicles (EVs) present in airway fluid carrying the long non-coding RNA MALAT-1, and the downstream implication of transcriptional firing and mitochondrial rewrting, with histone deacetylase 11 (HDAC11) and cMyc as key modulators. Remarkably, we show that GRIM neutrophils produced in vitro secrete secondary EVs that can in turn induce GRIM adaptation in a new wave of neutrophils. Also, we show that siRNA against MALAT-1 and HDAC11 and inhibition of HDAC11 by the small molecule drug SIS17 normalize granule release and bacterial killing by GRIM neutrophils, as well as their release of secondary EVs. Our objective is to fully characterize mechanisms of GRIM adaptation in CF airway neutrophils. Our central hypothesis is that chronic CF airway inflammation is a feed-forward process in which resident GRIM neutrophils release EVs that induce the GRIM fate in newly recruited neutrophils. This study introduces a new and innovative basic paradigm of neutrophil-driven human lung inflammation with key roles for EVs, MALAT-1, HDAC11 and cMyc, and innovative methods to deconstruct this paradigm (model for mass production of lung-recruited neutrophils, optimized tools to characterize lung EVs, and drug- / RNA / siRNA interventions targeted at neutrophils). We expect this study to yield transformative findings in our basic understanding and ability to treat chronic neutrophilic inflammation and infection in CF and other diseases.