Huang Weilan, Ling Shiqi, Jia Xiuhua, Lin Binwu, Huang Xi, Zhong Jing, Li Weihua, Lin Xiaolei, Sun Yifang, Yuan Jin
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center of Sun Yat-sen University, Guangzhou, China; Physical Examination Center, The Third Affiliated Hospital of Sun Yat-Sen University-Lingnan Hospital, Guangzhou, China.
Department of Ophthalmology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
PLoS One. 2014 Dec 2;9(12):e114386. doi: 10.1371/journal.pone.0114386. eCollection 2014.
To investigate the efficacy and mechanism of tacrolimus(FK506), which is a novel macrolide immunosuppressant, in inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) expression in a murine keratitis model induced by Aspergillus fumigatus.
TREM-1 was detected in 11 fungus-infected human corneas by quantitative real-time PCR (qRT-PCR). RAW264.7 macrophages were divided into four groups, which received treatment with zymosan (100 µg/ml), zymosan (100 µg/ml) + mTREM-1/Fc protein (1 µg/ml), or zymosan (100 µg/ml) + FK506 (20 µM) or negative-control treatment. After this treatment, the expression of TREM-1, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) was assayed using qRT-PCR and ELISA. The mouse model of fungal keratitis was created by intrastromal injection with Aspergillus fumigatus, and the mice were divided into 2 groups: group A received vehicle eye drops 4 times each day, and group B received 4 doses of FK506 eye drops each day. Corneal damage was evaluated by clinical scoring and histologic examination,and myeloperoxidase (MPO) protein levels were also detected by ELISA. The expression of TREM-1, IL-1β and TNFα was then determined at different time points using qRT-PCR and ELISA.
TREM-1 expression dramatically increased in the human corneas with fungal keratitis. In contrast, FK506 reduced the expression of TREM-1, IL-1β and TNFα in RAW264.7 macrophages stimulated with zymosan. In the mouse model, at day 1 post-infection, the corneal score of the FK506-treated group was lower than that of the control, and polymorphonuclear neutrophil (PMN) infiltration was diminished. TREM-1, IL-1β and TNFα expression was significantly reduced at the same time point. However, the statistically significant differences in cytokine expression, clinical scores and infiltration disappeared at 5 days post-infection.
FK506 may inhibit the inflammation induced by fungi and alleviate the severity of corneal damage at an early stage of fungal keratitis by downregulating TREM-1 expression.
研究新型大环内酯类免疫抑制剂他克莫司（FK506）在烟曲霉诱导的小鼠角膜炎模型中抑制髓系细胞触发受体-1（TREM-1）表达的疗效及机制。
采用定量实时PCR（qRT-PCR）检测11例真菌性感染人角膜中的TREM-1。将RAW264.7巨噬细胞分为四组，分别用酵母聚糖（100μg/ml）、酵母聚糖（100μg/ml）+mTREM-1/Fc蛋白（1μg/ml）、酵母聚糖（100μg/ml）+FK506（20μM）处理或给予阴性对照处理。处理后，采用qRT-PCR和ELISA检测TREM-1、白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNFα）的表达。通过基质内注射烟曲霉建立真菌性角膜炎小鼠模型，将小鼠分为2组：A组每天滴注赋形剂眼药水4次，B组每天滴注4剂FK506眼药水。通过临床评分和组织学检查评估角膜损伤，并采用ELISA检测髓过氧化物酶（MPO）蛋白水平。然后在不同时间点用qRT-PCR和ELISA检测TREM-1、IL-1β和TNFα的表达。
真菌性角膜炎患者角膜中TREM-1表达显著增加。相反，FK506降低了酵母聚糖刺激的RAW264.7巨噬细胞中TREM-1、IL-1β和TNFα的表达。在小鼠模型中，感染后第1天，FK506治疗组的角膜评分低于对照组，多形核中性粒细胞（PMN）浸润减少。在同一时间点，TREM-1、IL-1β和TNFα表达显著降低。然而，感染后5天时，细胞因子表达、临床评分和浸润的统计学显著差异消失。
FK506可能通过下调TREM-1表达抑制真菌诱导的炎症，并在真菌性角膜炎早期减轻角膜损伤的严重程度。
