Purpose
To investigate whether tacrolimus suppresses triggering receptor expressed on myeloid cells-1(TREM-1) expression in fungal keratitis and its effects.
Methods
RAW264.7 macrophages were devided into four groups: group i received zymosan(100ug/ml),group ii received zymosan (100ug/ml)+mTREM-1/Fc(20uM), group iii received zymosan(100ug/ml)+FK506(20uM), and group iv was the control and received no treatment. The expression of TREM-1,Interleukin-1β(IL-1β) and tumor necrosis factor alpha(TNFα) were assayed at 0h,4h,8h,12h,24h and 48h after zymosan treatment using real-time reverse transcriptase polymerase chain reaction (RT-PCR). The protein levels of sTREM-1,IL-1β,and TNFα were tested using ELSIA. An Aspergillus Fumigatus spore preparation (AFSP) was injected intrastromally to establish the C57BL/6J(B6) mouse model of fungal keratitis.Then, the mice were randomized into 2 groups: group A received four doses of FK506 eye drops each day, and group B received four doses of menstruum eye drops each day. The expression of TREM-1,IL-1β and TNFα was determined using real-time RT-PCR and ELISA at different time points. Myeloperoxidase(MPO) protein levels were confirmed by ELISA. Corneal damage was evaluated by clinical scores and histologic examination.
Results
FK506 reduced the expression of TREM-1,IL-1β and TNF-α in RAW264.7 macrophages stimulated by zymosan. TREM-1 expression was significantly reduced in FK506-treated mouse corneas with fungal keratitis at 1 day and 3 days post-infection (P<0.05). There was no change detected at 5 days post-infection between the two groups. The expression levels of IL-1β and TNF-α presented the same trend as TREM-1 at 1 day post infection (P<0.05). The clinical score of group A at 1 day p.i.was 2.8 ±0.63, and the clinical score of group B was 2±0.66 (P<0.05).There was no statistically significant difference between the two groups at 3 and 5 days p.i. Hematoxylin-eosin(HE) staining and MPO ELISA data indicated that polymorphonuclear neutrophil(PMN) infiltration was diminished in group A at 1 day p.i.(P<0.001).The CFU data indicated a significant decrease in fungal viability at 1 and 3 days p.i. in group A (P<0.05).
Conclusions
FK506 suppressed the inflammatory response in a fungal keratitis mouse model by down-regulating TREM-1,IL-1β and TNF-α expression and reducing the infiltration of PMNs.
Keywords: 480 cornea: basic science •
557 inflammation •
573 keratitis