Formation Of NETs And Influence Of Glucocorticoid On NETs Infungal Keratitis

Background and ObjectionFungal keratitis is a severe infectious diseases carrying the risk of vision impairment,especially in developing countries.In our country,the blindness rate of fungal keratitis has been the top in those infected keratitis.The fungal keratitis was first reported by Dr.Leber in 1897.In recent years, due to the widespread use of broad-spectrum antibiotics and glucocorticoid and the long-term use of immunosuppressive agents and the increase use of contact lens the incidence of fungal keratitis shows a increasing tendency.Additionally,the sensitivity of the detection method have been greatly improved,that lead to more patients confirmed.Trauma has been considered as the a major cause of fungal keratitis,especially the agriculture trauma.Different countries have different pathogens.In the developed and colder areas the major pathogen is candida albicans.Fusarium and Aspergillus is common in developing areas and hotter areas.Fungal keratitis can lead to serious corneal ulceration or corneal perforation, endophthalmitis and endanger the whole eye.However,recently there is a lack of broad-spectrum, high efficiency, low toxicity anti fungal drugs.Many studies confirm that glucocorticoid drugs can aggravate the condition of fungal keratitis.But the exact mechanism is unclear.The innate immune system is the first line of defense against invading pathogens,limiting the spread of pathogens in the early stages of infection.Neutrophils as key members of the innate immunity plays an important role in this process.Recently,a novel bactericidal mechanism of neutrophils was discovered named neutrophil extracellular traps (NETs) and the formation of NETs was named Netosis.Netosis is a recently described specific type of neutrophil death,different from necrosis and apoptosis.Upon contact with various bacteria or fungi as well as with activated platelets or under the influence of numerous inflammatory stimuli, neutrophils releasing their chromatin to the extracellular medium associated with histones and granular and cytoplasmic proteins.The DNA scaffold entraps the pathogens and limits their spread,then the attached proteins will kill these pathogens.Many researchers have found the formation of NETs in many inflammatory and infectious diseases,suggesting it plays an important role in inflammation and infection diseases.However,many investigators also find the overproduction of NETs will damage the tissues.Due to lack of corneal blood vessels,how the NETs in cornea metabolism need more research.Considering glucocorticoid has strong anti-inflammatory and immune inhibitory effect and the NETs is the important antibacterial mechanism of neutrophils.Whether glucocorticoid by influencing the formation of NETs affect fungal keratitis condition need more studies.If we can reveal some connection between glucocorticoids and the NETs,that will guide the rational use of glucocorticoids in clinical.By modulating the formation of NETs,we can achieve the goal of efficient elimination of pathogens with less injury to the tissues.MethodsThe animal models of fungal keratitis were established by corneal surface lens method. C57BL/6 mice were randomly divided into non-intervention group,dexamethasone treated group and injury control group.Candida albicans were inoculated in non-intervention group and dexamethasone treated group and normal saline were inoculated in injury control group.Take out stitches in 48 hours and then dexamethasone treated group and injury control group were treated with 0.1% dexamethasone drops and non-intervention group were treated with normal saline for 3 days.The influence of glucocorticoid on fungal keratitis were observed at 1,3,5,7,10 days after infection.All the experimental data were processed by SPSS 17.0.All reported P values are evaluated by 2 sides.Nonparametric test of Mann-Whitney U test was used for statistical analysis.ResultsThe infected corneas from non-intervention group and dexamethasone treated group showed typical fungal keratitis.Compared with non-intervention group during 10 days after innoculation, the dexamethasone-treated group showed more severe fungal keratitis and the course was extended.Keratitis in non-intervention group started to resolved around days 7～10 but dexamethasone-treated group still showed severe fungal keratitis in the 10th day by disease presentation, histology and pathogen burden assay. The corneas form dexamethasone-treated group contained more pathogens than non-intervention group during the measured period(10 days).Under the influence of dexamethasone,the hyphae of fungus in cornea grow longer approaching the corneal descemet membrane.However,there were less infiltration of neutrophils in the cornea form dexamethasone-treated group.The scanning electron microscope showed NETs entrap the Candida albicans and limit their spread.After the dexamethasone treatment, more hyphae of fungus grew in the ulcer lesions but less neutrophils infiltrated and less NETs formed-Compared with non-intervention group,the NETs in dexamethasone-treated group was sparse and less protein attached.Conclusions1.Neutrophils form the NETs to entrap the Fungi in fungal keratitis.In the course of against infection,neutrophils by phagocytosis and formation of NETs fight against pathogens.2.Glucocorticoids can accelerate the growth of Candida albicans and enhanced the invasion of Candida albicans.However,glucocorticoids reduce the infiltration of neutrophils and the formation of NETs.After glucocorticoids treatment, bactericidal capacity of NETs was also weakened.