Immune Response And Mechanism Of IFN-? In The Administration For Keratomycosis

Objective:To investigate the immune response and the possible mechanism of IFN-? in the treatment of fungal keratitis in mice.Method:Experimental murine Fungal Keratitis was induced by epikeratophakia with the aid of corneal epithelium erasion with a minor modification using Fusarium solani spores. Mice were divided into three groups,group A: tropical PBS four times daily post-infection, group B: tropical IFN-?(2.5×105U/m L)four times daily post-infection. One, three, five, seven, and ten days post-infection, the corneal lesions and inflammatory responses were observed by slit-lamp, clinical scores and fungal load were measured regularly, immunofluorescence staining were performed to evaluate macrophages and CD4+ cells in infected cornea distribution. and the expressions of the six inflammatory cytokines, macrophage Migration Inhibitory Factor(MIF), macrophage inflammatory protein-2(MIP-2), interleukin-4(IL-4), interleukin-10(IL-10), interleukin-6(IL-6) and interferon-?(IFN-?), in the infected corneas were determined using reverse transcription polymerase chain reaction(RT–PCR), and enzyme-linked immunosorbent assay(ELISA) was used to detect macrophage Migration Inhibitory Factor(MIF) in infected cornea.Result: Dropping tropical IFN ? can significantly reduce pathological changes of the fungal infected corneal tissue, decrease clinical scores, shorten the duration of fungal keratitis. The clinical scores of group B(tropical IFN ?) in five, seven, and ten days post-infection were statistically significant difference compared with the group A(p<0.05). According to the fungal load results of the two groups in the five time points, fungal load results in cornea are present the overall downward trend over time. And corneal fungal load in IFN ? treated group(group B) is less than the group A in the fifth day after fungal infection(P < 0.05), indicating that interferon ? eye drops could shorten the course of the fungal keratitis. Specifically, corneal fungal load did not see obvious differences between two groups on the first day, in the third day after inoculation, corneal fungal load of group B was less than group A, but have no obvious statistical difference; in fifth day fungal loads of group B was significantly less than group A, and statistical analysis of p < 0.05, suggesting that the difference was statistically significant. There was no fungus detected in homogenate cornea seven and ten days post-infection. Macrophages in cornea of group B expressed highly than group A in early stage, namely IFN ? eye drops could activate the body's immune, speed up the process of eliminating fungal pathogens. Immunofluorescence showed that group B has visible F4/80(red fluorescence) expression in all five observational time point(one, three, five, seven and ten days post-infection), and expressed higher than group A, while in five seven and ten days, the F4/80 express of group A was not obvious. CD4+ cells(green fluorescence) expression in group A was not obvious in one and three days, while group B expressed weak fluorescence in one, three, five, seven and ten days post-infection. Real-Time PCR results showed that the concentration of IL-4 m RNA in group B was significantly lower than the group A(differences were statistically significant in each time points, p < 0.05), which slowly decline over time, while the concentration of IL-4 in group A raised from the first day to seventh day, which dropped substantially on the tenth day. The tendency of the concentration of IL-10 and IFN ? seem to be roughly same, the tendency shows first increase and then decrease along with the development of the course, but in group B reached its peak in fifth day, and group A reached their peak in seventh day. Besides the concentrations of IL-10 and IFN ? m RNA in group B were significantly lower than group A in 7th day which differences were statistically significant(p < 0.05). Group B was clearly presented the trend of rising and falling comparing to group A concerning to the concentration of IL-12, which reached its peak in the fifth day, and much higher in 1th, 3th and 5th days(differences were statistically significant, p< 0.01). MIP-2 expression became less and less over time in two groups, but in group B expressed higher amount than group A at all time points(differences were statistically significant, p< 0.05). When dropped with IFN ?, the quantity of MIF expression is significantly higher than group A(difference was statistically significant at 1th and 5th days, P < 0.05).The ratio of IL-4 /IL-12 m RNA presented a significantly lower proportion in fungal infected cornea after IFN ? eye dropped(differences were statistically significant at 1, 3, 5, 7 and 10 days, P < 0.05), indicating that IFN ? can make the body's immune deviate Th1 direction. ELISA result showed that MIF protein expressed heigher in group A when compaired to group B in 1, 3, 5 and 10 days(differences were statistically significant at 3 and 7 days, P < 0.01).Conclusion: This experimental experiment showed that treatment with IFN ? eye drops that help the body clear cornea fungi more quickly, increase the gather of macrophages in the cornea, make the body's immune to Th1 direction deviation, and shorten the duration of fungal keratitis in mice fungal keratitis.