Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.
Department of Ophthalmology, Qingdao Central Hospital, Qingdao, Shandong Province, China.
Invest Ophthalmol Vis Sci. 2024 Jul 1;65(8):4. doi: 10.1167/iovs.65.8.4.
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.
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.
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.
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.
本研究旨在探讨微管相关蛋白轻链 3（LC3）相关噬菌作用（LAP）在烟曲霉菌角膜炎免疫反应中的作用和机制。
使用透射电子显微镜（TEM）观察健康或烟曲霉菌感染的人及 C57BL/6 小鼠角膜中单膜噬菌囊的形成。用 Rubicon siRNA（si-Rubicon）阻断 Rubicon 表达。用 si-Rubicon 和乱序 siRNA 预处理 RAW 264.7 细胞或小鼠角膜，然后用烟曲霉菌感染。用 Dectin-1 抗体或 Dectin-1 过表达质粒预处理 RAW 264.7 细胞，然后用烟曲霉菌刺激。用流式细胞术标记正常和感染角膜中的巨噬细胞。在烟曲霉菌角膜炎小鼠中，用临床评分评估疾病严重程度。我们用慢病毒技术将 GV348-Ubi-GFP-LC3-II-SV40-Puro 慢病毒转入小鼠角膜。用荧光显微镜观察角膜切片中的 GFP-LC3 融合蛋白。用实时 PCR（RT-PCR）和 ELISA 检测炎症因子 IL-6、IL-1β 和 IL-10 的 mRNA 和蛋白表达。用 Western blot 或免疫荧光法检测 LAP 相关蛋白 Rubicon、ATG-7、Beclin-1 和 LC3-II 的表达。
TEM 观察到烟曲霉菌角膜炎患者和小鼠角膜中巨噬细胞内单膜噬菌囊的积累。FCM 分析结果显示，烟曲霉菌感染后小鼠角膜中巨噬细胞数量明显增加。烟曲霉菌感染后，小鼠和 RAW 264.7 细胞中 LAP 相关蛋白表达显著升高。Rubicon siRNA 处理可升高小鼠临床评分。烟曲霉菌角膜炎小鼠中，Rubicon siRNA 处理组较对照组，IL-6 和 IL-1β 表达显著升高，IL-10 和 LC3-II 表达显著降低。在 RAW 264.7 细胞中，Dectin-1 过表达质粒上调 LAP 相关蛋白表达，该过程可被 Dectin-1 抗体显著抑制。
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 通过 Dectin-1 信号通路在烟曲霉菌角膜炎中被调控。
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