| Purpose:To investigate triggering receptor expressed on myeloid cells-1 (TREM-1) expression in corneal epithelial cells infected by fungus and evaluate its role.Methods:Patients and rat models of fungal keratitis (FK) were used for in vivo experiments. The telomerase-immertalized human corneal epithelial cells (THCEs) were cultured for in vitro experiments. (1) To determine whether TREM-1 mediates corneal inflammation after fungal infection, patients with FK at the Department of Ophthalmology (The Affiliated Hospital of Qingdao University) from September 2012 to October 2013 were included. A total of 25 first-visit patients were divided into three groups according to keratomycosis severity, which was scored visually with the aid of a slit lamp:mild group, moderate group and severe group. Controls were normal corneal tissues remaining after corneal transplantation. Corneal epithelial scrapings were collected and the mRNA of TREM-1 was analyzed using Real-time PCR. Wistar rats were randomly divided into control group, sham group and FK group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8,16, 24,48 and 72 hours after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1 through quantitative RT-PCR, western blot analysis and immunofluorescence technique. The correlation between FK inflammation and expression of TREM-1 was also analyzed. (2) To determine the effect of A. fumigatus infection on TREM-1 expression in THCEs, THCEs were treated with spores and mycelium of A. fumigatus respectively. TREM-1 expression was evaluated by Real-time PCR and western blot analysis. To examine the function of TREM-1 in inflammatory cytokine production, antagonist LP17 peptide (LQVTDSGLYRCVIYHPP) of TREM-1 was added to THCEs at 100 ng/mL, and then the cells were subjected to A. fumigatus challenge for 4 h and 8 h. (3) The interaction between TREM-1 and Toll-like receptor-4 (TLR-4) was investigated by use of LP17, CLI-095 (an inhibitor of TLR-4 signaling) and ST2825 (an inhibitor of MyD88). LP17, CLI-095 and ST2825 were respectively added to THCEs that were cultured in 12-well flat-bottom plates, then to A. fumigatus spore challenge for 8 h. TREM-1, TLR-4 and MyD88 protein expressions in THCEs were determined by western blot analysis.Results:(1) PCR data showed that, compared with controls, TREM-1 was significantly enhanced in the human corneal epithelium with fungal infection (P< 0.05). Moreover, the expression levels of TREM-1 increased with increasing keratomycosis severity. In rat models of FK, corneal inflammation scores increased with time after fungal infection (P< 0.05). The inflammation scores in FK group were obviously higher than those in sham group on the whole (P< 0.05). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h (P< 0.001, compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24h and 48h after fungal infection (P< 0.05, compared with control group). Immunofluorescence technique showed that TREM-1 mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1 expression in FK (r=0.942, P< 0.001). (2) The expression of TREM-1 mRNA in THCEs infected with spores of A. fumigatus was higher than that in THCEs infected with mycelium of A. fumigatus (P< 0.05). Western blotting confirmed that A. fumigatus infection increased TREM-1 protein expression at 4,8, and 16 h and the expression level peaked at 8 h (P< 0.05). The upregulation of TREM-1 expression induced by A. fumigatus can be inhibited by LP at the concentrations of lOng/ml,50ng/ml and 100ng/ml in a concentration dependent manner (P< 0.05). RT-PCR data demonstrated that incubation with A. fumigatus led to upregulation of TNF-α, IL-1β, IL-6, and IL-8 compared with controls at 4 h and 8 h. However, LP17-induced blockade of TREM-1 significantly reduced the expression levels of the above inflammatory cytokines mRNA at 4 h and 8 h post-infection (P< 0.05). (3) LP17 significantly inhibited A. fumigatus induced upregulation of TREM-1, TLR-4 and MyD88. Treatment with CLI-095 also partially blocked A. fumigatus-induced upregulation of TREM-1 and TLR-4. Moreover, TREM-1 can also be inhibited by the inhibitor of MyD88-ST2825, potently. RT-PCR data showed that LP17/CLI095 inhibited A. fumigatus-induced upregulation of TNF-a, IL-1β, IL-6, and IL-8. There was no statistical difference between TREM-1 inhibition and TLR-4 inhibition (P> 0.05). The combined blockade of TREM-1 and TLR-4 significantly reduced the stimulatory secretion compared with TREM-1 or TLR-4 inhibition alone (P< 0.05).Conclusions:(1) TREM-1 may contribute to amplify the inflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses. (2) TREM-1 plays critical roles in fungal infection, and targeting it may represent a novel therapeutic strategy for patients with FK. (3) The cross-talk occurs between TREM-1 and TLR-4 in cornea fungal infection. (4) The expression of TREM-1 in response to A. fumigatus is MyD88-dependent.
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