The Role Of Neutrophil Extracellular Traps In Fungal Keratitis Of Mice In Early Stage

Background Fungal keratitis is a kind of corneal infection which is caused by invasion of fungi. It can cause high rate of blindness and mainly happened among the farmers in China. The pathogenesis of fungal keratitis in China is closely related to the plant trauma, and the pathogenic fungi are mainly filamentous fungi such as fusarium and aspergillus. Researches suggest that neutrophils are the main immune cells in the early stage of corneal fungal infection. In addition to the classic phagocytosis, degranulation and the respiratory burst mechanism, neutrophil can kill pathogenic microorganism by neutrophil extracellular trap. Studies have shown that NETs can be induced by both aspergillus and candida albicans in vitro. NETs resistance to fungal infection in vivo mainly concentrated in pulmonary aspergillosis and fungal infection in skin and oral. However, the role of NETs in fungal keratitis caused by fusarium solani has not yet been reported. Therefore, to investigate the role of neutrophil extracellular traps in early stage of mice fungal keratitis will provide theory basis or potential drug targets for the therapy of fungal keratitis.Objective To investigate the role of neutrophil extracellular traps in early stage of mice fungal keratitis.Materials and methods Twenty-eight male C57BL/6J mice of SPF grade were used in the experiment. All mice were 8-12 weeks of age. Mouse corneas were examined with a slit lamp before operation. C57BL/6J mice without any eye disease were randomly divided into fungal keratitis model group and sham group. After operation, mice were randomly divided into 0h, 12 h, 24 h, 36 h sub-groups and each sub-group with 8 eyes(n = 8). Mice with normal corneas were used as 0h sub-group. For fungal keratitis model group, a cross scratch was created in the central cornea which was marked with 1-mm trephine and mice corneas were inoculated with Fusarium solani. For sham group, a cross scratch was created in the central cornea without inoculation with fungi. Pictures of each cornea were taken by a stereomicroscope and the cornea severity was evaluated by clinical score at each time points mentioned above using a slit lamp. Mice were sacrificed at 0h, 12 h, 24 h and 36 h respectively. Two corneas were excised and fixed in 2.5% glutaraldehyde for NETs detection by transmission electron microscope(TEM). Six corneas were collected to perform corneal whole mount at each time points. Histone, neutrophil elastase, fungi and nucleus of cornea were labeled with anti-Histone H1 antibody, anti-neutrophil elastase antibody, white calcium fluorescence(Calcofluor White, CFW) and DAPI respectively using immunofluorescence staining. The whole mount were divided into limbal, periphery, paracentral and central cornea and each field was scanned using Z-stack mode by two-photon confocal scanning microscope. Clinical score, NETs assay and volume of fungi were determined and analyzed using SPSS16.0 software.Results Normal corneas were transparent with intact epithelium. The cornea wound of sham group and model group were unhealed and with mild opacity at 12 h after operation. The wound was almost healed in sham group at 24 h. However, corneal opacity was severe and typical clinical manifestations of fungal keratitis were seen on the cornea in model group at the same time points. The cornea wound was completely healed in sham group at 36 h, but the disease progressed rapidly and corneal lesions were more severe with opacity and abundant hypopyon in model group. The significant differences were seen in clinical scores at different time points in sham group(c2=17.743, P=0.000<0.05). The clinical scores of corneal lesions were significantly high at 12 h and 24 h compared with 0h(P=0.001<0.05). There were no significant difference between 36 h and 0h in sham group(P=0.138>0.05) and also no difference between 24 h and 12h(P=1.000>0.05). There were significant differences at both 12 h and 24 h compared with 36 h in sham group(P=0.019<0.05). The clinical scores increased gradually and there were significant differences at different time points in model group(c2 =21.129, P=0.000<0.05). The clinical scores of corneal lesions were significantly high at each time points compared with 0h(P=0.001, 0.002<0.05).There were significant differences at 24 h and 36 h compared with 12h(P=0.002, 0.003<0.05). However, no difference was seen between 24 h and 36h(P=0.057>0.05). There was no difference between sham group and model group at 12 h after operation(P=0.317>0.05), but significant differences were seen between groups at 24 h and 36h(P=0.002, 0.003<0.05). NETs structure was undetected in normal cornea. There was a small amount of neutrophil infiltration in the central cornea and no typical NETs structure in sham group at each time points. A small amount of NETs was seen in the central cornea at 12 h in model group. There were a lot of typical NETs trapping fungi in the central cornea visualized by immunofluorescence staining and transmission electron microscope at 24 h in model group. Fungal cell wall were broken and the cellular organelle were lysed. NETs were also detected in paracentral and periphery and mainly distributed in stroma. The interaction between NETs and fungal hyphae were evident in central and paracentral cornea and hyphae were destroyed obviously at 36 h in model group. The infiltrated neutrophils formed a ring around hyphae to restrict its spreading to periphery cornea. NETs assay increased from 12 h to 24 h and reached peak at 24 h and then decreased at 36 h. There were significant differences in NETs assay at different time points in model group(c2 =12.331, P =0.002<0.05). The NETs assay of infected corneas were significantly different between 24 h and 12h(P=0.004<0.05). NETs decreased at 36 h and there were significant differences between 36 h and 24h(P=0.006<0.05). However, there was no difference between 36 h and 12h(P= 0.077>0.05). The volume of fungi increased from 12 h to 36 h in model group and there were significant differences at each time points(Ftime=28.598, P=0.000<0.05). The fungal volume significantly increased at 24 h compared with 12h(P=0.025<0.05). There were significant differences at 36 h compared with 12 h and 24h(P=0.001, 0.020<0.05). The volume of fungi had no correlation with NETs assay in model group(r=0.054, P=0.832>0.05). However, the clinical score was positive correlated with NETs assay(r=0.514, P=0.029<0.05) and also with fungi volume(r=0.828, P=0.000<0.05).Conclusions 1. NETs can be induced by Fusarium solani in fungal keratitis of mice in early stage and NETs assay reach peak at 24 h. 2. Both NETs and fungi affect the severity of corneal lesions in fungal keratitis in early stage.