Outer membrane vesicles in macrophage inflammation and pyroptosis

项目总结 巨噬细胞等先天免疫细胞通过识别病原体相关物质来检测病原体 上显示的分子图案(PAMP)，如脂多糖(LPS，也称为内毒素) 革兰氏阴性细菌的表面。Toll样识别细胞外和内体内的内毒素分子 另一方面，胞浆内的内毒素是通过caspase-11(在小鼠体内)和caspase-4/5检测到的 (在人类身上)。有趣的是，最近发现外膜囊泡(OMV)由 胞外革兰氏阴性菌可将内毒素和其他细菌分子送入肺炎衣原体的胞浆 巨噬细胞。OMVS首先通过内吞作用进入巨噬细胞，然后转位 细菌分子穿过内吞体膜进入胞浆引发下垂，引起炎性反应 一种细胞死亡的形式。OMV引发的上睑下垂的分子基础仍然知之甚少。在……里面 特别是，OMV是如何结合和进入巨噬细胞的，以及OMV是如何穿过 内吞体膜。这项拟议研究的目标是通过以下方式解决这些关键问题 利用无偏全基因组CRISPR基因对OMV诱导的上睑下垂进行遗传学解剖 屏幕上。在我们的初步研究中，我们建立了检测OMV诱导的上睑下垂的方法 巨噬细胞。此外，我们开发了工具，用来从基因上剖析复杂的哺乳动物通路 全基因组CRISPR筛选。在这里，我们将利用这些系统的战略优势来剖析OMV-- 使用无偏全基因组CRISPR基因筛查在巨噬细胞中诱导下垂。我们会 然后在集合和目标二级筛查中验证已识别的基因。最后，我们将进一步 鉴定在原代人类巨噬细胞筛选中确定的选定基因。这些研究将 提供OMV诱导的上睑下垂的基因组规模的观点，并将扩展我们对炎症的知识 病原菌-寄主相互作用中的反应。从这项试点研究中收集到的见解将有助于 开发更安全、更有效的策略来治疗病原体感染和炎症性疾病。 此外，我们预计这些发现将指导使用OMV的药物输送车辆的工程设计。

PROJECT SUMMARY Innate immune cells such as macrophages detect pathogens by recognizing pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS, also known as endotoxin) displayed on the surface of Gram-negative bacteria. Extracellular and endosomal LPS molecules are recognized by Toll-like receptor 4. Cytosolic LPS, on the other hand, is detected by caspase-11 (in mice) and caspase-4/5 (in humans). Interestingly, it was recently found that outer membrane vesicles (OMVs) released by extracellular Gram-negative bacteria can deliver LPS and other bacterial molecules to the cytosol of macrophages. OMVs first enter macrophages through endocytosis, followed by the translocation of bacterial molecules across the endosome membrane into the cytosol to trigger pyroptosis, an inflammatory form of cell death. The molecular basis of OMV-triggered pyroptosis remains poorly understood. In particular, it is unknown how OMVs bind and enter macrophages, and how OMVs crosses the endosome membrane. The goal of this proposed research is to address these key questions by genetically dissecting OMV-induced pyroptosis using an unbiased genome-wide CRISPR genetic screen. In our preliminary studies, we established assays to measure OMV-induced pyroptosis in macrophages. Moreover, we developed tools to genetically dissect complex mammalian pathways using genome-wide CRISPR screens. Here, we will take strategic advantage of these systems to dissect OMV- induced pyroptosis in macrophages using an unbiased genome-wide CRISPR genetic screen. We will then validate the identified genes in pooled and targeted secondary screens. Finally, we will further characterize selected genes identified in the screens in primary human macrophages. These studies will provide a genome-scale view of OMV-induced pyroptosis and will expand our knowledge of inflammatory responses in pathogen-host interactions. Insights gleaned from this pilot study will facilitate the development of safer and more effective strategies to treat pathogen infection and inflammatory diseases. Moreover, we anticipate that these findings will guide the engineering of drug delivery vehicles using OMVs.