Tacrolimus (FK506) Suppresses TREM-1 Expression at an Early but Not at a Late Stage in a Murine Model of Fungal Keratitis

Purpose

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.

Method

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.

Results

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.

Conclusions

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.