PDF(Pubmed)
Abstract：
UNASSIGNED: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis.
UNASSIGNED: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence.
UNASSIGNED: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody.
UNASSIGNED: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.
UNASSIGNED: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence.
UNASSIGNED: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody.
UNASSIGNED: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.
摘要：
■本研究的目的是研究微管相关蛋白轻链3（LC3）相关吞噬作用（LAP）在对烟曲霉的免疫反应中的作用和机制（A.烟曲霉）角膜炎。
■使用透射电子显微镜（TEM）在健康或烟曲霉感染的人和C57BL/6小鼠的角膜中观察到单膜吞噬体的形成。使用RubiconsiRNA(si-Rubicon)来阻断Rubicon表达。使用或不使用si-Rubicon和乱序siRNA预处理的烟曲霉感染RAW264.7细胞或小鼠角膜。用Dectin-1抗体或Dectin-1过表达的质粒预处理RAW264.7细胞,然后用烟曲霉刺激。流式细胞术用于标记小鼠正常和感染角膜中的巨噬细胞。在患有烟曲霉角膜炎的小鼠中，使用临床评分评估疾病的严重程度.我们使用慢病毒技术将GV348-Ubi-GFP-LC3-II-SV40-Puro慢病毒转移到小鼠角膜中。使用荧光显微镜在角膜切片中观察GFP-LC3融合蛋白。检测炎症因子IL-6、IL-1β的mRNA和蛋白表达,和IL-10使用实时PCR(RT-PCR)和ELISA。我们检测到LAP相关蛋白Rubicon的表达，使用蛋白质印迹或免疫荧光法,ATG-7、Beclin-1和LC3-II。
■使用TEM在患有烟曲霉角膜炎的患者和小鼠的角膜中观察到巨噬细胞内单膜吞噬体的积累。流式细胞术（FCM）分析结果表明，感染烟曲霉后，小鼠角膜中的巨噬细胞数量显着增加。用烟曲霉感染后，小鼠角膜和RAW264.7细胞中LAP相关蛋白显着升高。si-Rubicon治疗提高了小鼠的临床评分。在烟曲霉角膜炎小鼠中，与对照组相比,si-Rubicon处理组显示显著更高的IL-6和IL-1β表达和更低的IL-10和LC3-II表达。在RAW264.7单元格中，用Dectin-1过表达的质粒处理上调了LAP相关蛋白的表达，Dectin-1抗体显著抑制的过程。
■LAP参与真菌性角膜炎（FK）的抗炎免疫过程，并发挥抗炎作用。LAP在烟曲霉角膜炎中通过Dectin-1信号通路调节。
■使用透射电子显微镜（TEM）在健康或烟曲霉感染的人和C57BL/6小鼠的角膜中观察到单膜吞噬体的形成。使用RubiconsiRNA(si-Rubicon)来阻断Rubicon表达。使用或不使用si-Rubicon和乱序siRNA预处理的烟曲霉感染RAW264.7细胞或小鼠角膜。用Dectin-1抗体或Dectin-1过表达的质粒预处理RAW264.7细胞,然后用烟曲霉刺激。流式细胞术用于标记小鼠正常和感染角膜中的巨噬细胞。在患有烟曲霉角膜炎的小鼠中，使用临床评分评估疾病的严重程度.我们使用慢病毒技术将GV348-Ubi-GFP-LC3-II-SV40-Puro慢病毒转移到小鼠角膜中。使用荧光显微镜在角膜切片中观察GFP-LC3融合蛋白。检测炎症因子IL-6、IL-1β的mRNA和蛋白表达,和IL-10使用实时PCR(RT-PCR)和ELISA。我们检测到LAP相关蛋白Rubicon的表达，使用蛋白质印迹或免疫荧光法,ATG-7、Beclin-1和LC3-II。
■使用TEM在患有烟曲霉角膜炎的患者和小鼠的角膜中观察到巨噬细胞内单膜吞噬体的积累。流式细胞术（FCM）分析结果表明，感染烟曲霉后，小鼠角膜中的巨噬细胞数量显着增加。用烟曲霉感染后，小鼠角膜和RAW264.7细胞中LAP相关蛋白显着升高。si-Rubicon治疗提高了小鼠的临床评分。在烟曲霉角膜炎小鼠中，与对照组相比,si-Rubicon处理组显示显著更高的IL-6和IL-1β表达和更低的IL-10和LC3-II表达。在RAW264.7单元格中，用Dectin-1过表达的质粒处理上调了LAP相关蛋白的表达，Dectin-1抗体显著抑制的过程。
■LAP参与真菌性角膜炎（FK）的抗炎免疫过程，并发挥抗炎作用。LAP在烟曲霉角膜炎中通过Dectin-1信号通路调节。
