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Invest Ophthalmol Vis Sci. 2020 Feb 7;61(2):25. doi: 10.1167/iovs.61.2.25.
To determine the role of autophagy in the innate immune response to fungal keratitis (FK) caused by Aspergillus fumigatus infection.
Corneal samples obtained from patients and mice with FK were visualized via transmission electron microscopy (TEM). Autophagy-related proteins LC3B-II, Beclin-1, LAMP-1, and p62 in A. fumigatus-infected corneas of C57BL/6 mice were tested by Western blot. After treatment with autophagy inhibitors 3-methyladenine (3-MA), chloroquine (CQ), or inducer rapamycin, autophagy-related proteins were detected by Western blot. Corneas were photographed with slit lamp microscopy and pathological changes were observed by hematoxylin and eosin staining. Polymorphonuclear neutrophilic leukocytes (PMNs) were assessed by immunofluorescent staining and observed under TEM. The levels of CXCL-1, IL-1β, HMGB1, IL-18, TNF-α, and IL-10 were tested by reverse transcription polymerase chain reaction and Western blot. The quantification of fungal loads was detected and photographed.
The accumulation of autophagosomes in corneas of patients and mice with FK was observed with TEM. The expression of LC3B-II, Beclin-1, and LAMP-1 was elevated in corneas after fungal infection, whereas p62 was reduced. Treatment with 3-MA or CQ upregulated clinical scores, pathological changes, and the expression of CXCL-1, IL-1β, HMGB1, IL-18, and TNF-α except IL-10. The morphology of PMNs was changed and PMN recruitment was increased in mice corneas treated with 3-MA or CQ, whereas rapamycin reduced the inflammatory response to keratitis. These results were statistically significant.
A. fumigatus infection increases the expression of autophagy in corneas. Autophagy plays an anti-inflammatory role in the innate immune response to A. fumigatus keratitis.
确定自噬在烟曲霉感染引起的真菌性角膜炎（FK）固有免疫反应中的作用。
通过透射电子显微镜（TEM）观察 FK 患者和小鼠的角膜样本。通过 Western blot 检测烟曲霉感染的 C57BL/6 小鼠角膜中的自噬相关蛋白 LC3B-II、Beclin-1、LAMP-1 和 p62。用自噬抑制剂 3-甲基腺嘌呤（3-MA）、氯喹（CQ）或诱导剂雷帕霉素处理后，通过 Western blot 检测自噬相关蛋白。用裂隙灯显微镜拍摄角膜照片，用苏木精和伊红染色观察病理变化。通过免疫荧光染色评估多形核中性粒细胞（PMN），并在 TEM 下观察。通过逆转录聚合酶链反应和 Western blot 检测 CXCL-1、IL-1β、HMGB1、IL-18、TNF-α 和 IL-10 的水平。检测真菌负荷的定量并拍照。
TEM 观察到 FK 患者和小鼠角膜中自噬体的积累。真菌感染后角膜中 LC3B-II、Beclin-1 和 LAMP-1 的表达升高，而 p62 减少。用 3-MA 或 CQ 处理可上调临床评分、病理变化以及 CXCL-1、IL-1β、HMGB1、IL-18 和 TNF-α的表达（除了 IL-10）。用 3-MA 或 CQ 处理的小鼠角膜中 PMN 的形态发生变化，PMN 募集增加，而雷帕霉素可减轻角膜炎的炎症反应。这些结果具有统计学意义。
烟曲霉感染增加了角膜中自噬的表达。自噬在烟曲霉角膜炎固有免疫反应中发挥抗炎作用。
