Outer membrane vesicles in macrophage inflammation and pyroptosis

项目摘要 先天性免疫细胞如巨噬细胞通过识别病原体相关抗原来检测病原体。 分子模式（PAMPs），如脂多糖（LPS，也称为内毒素）显示在 革兰氏阴性菌表面。细胞外和内体LPS分子被Toll样蛋白识别， 受体4。另一方面，细胞溶质LPS通过caspase-11（在小鼠中）和caspase-4/5检测 (in人类）。有趣的是，最近发现，外膜囊泡（OMV）释放的 细胞外革兰氏阴性细菌可以将LPS和其他细菌分子递送到细胞质中， 巨噬细胞OMV首先通过内吞作用进入巨噬细胞，然后通过内吞作用转运到巨噬细胞。 细菌分子穿过内体膜进入胞质溶胶，引发细胞凋亡， 细胞死亡的形式。OMV引发的细胞凋亡的分子基础仍然知之甚少。在 特别是，OMV如何结合并进入巨噬细胞以及OMV如何穿过巨噬细胞是未知的。 内体膜这项研究的目标是解决这些关键问题， 使用无偏的全基因组CRISPR遗传分析方法， 屏幕在我们的初步研究中，我们建立了测定OMV诱导的细胞凋亡的方法， 巨噬细胞此外，我们还开发了一些工具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- 使用无偏的全基因组CRISPR遗传筛选在巨噬细胞中诱导细胞凋亡。我们将 然后在合并的和靶向的二次筛选中验证所鉴定的基因。最后，我们将进一步 表征在原代人巨噬细胞中筛选中鉴定的选定基因。这些研究将 提供了一个基因组规模的视图OMV诱导的pyroptosis，并将扩大我们的知识，炎症 病原体-宿主相互作用的反应。从这项试点研究中获得的见解将有助于 开发更安全和更有效的策略来治疗病原体感染和炎性疾病。 此外，我们预计这些发现将指导使用OMV的药物递送载体的工程。

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.