Expression And Mechanism Of CXCL10 In Aspergillus Fumigatus Keratitis

Purpose: To determine the expression and role of interferon-inducible protein 10(CXCL10)in Aspergillus fumigatus(A.fumigates)keratitis.Methods: In this experiment,the expression of CXCL10,the effects of glycyrrhizic acid(GA)pretreatment on the infection of A.fumigatus keratitis and the effects of the expression of relevant inflammatory cytokine were studied in the mouse model of A.fumigatus keratitis and immortalized human corneal epithelial cells(HCECs)infected by A.fumigatus.Based on that,the study was divided into three aspects: 1.After HCECs were stimulated by A.fumigatus conidia,the expression of CXCL10 m RNA was detected by Realtime PCR in normal immortalized HCECs and HCECs stimulated by A.fumigatus conidia for 4h、8h、10h、12h、16h and 20 h,the secretion of CXCL10 was detected by ELISA in normal immortalized HCECs and HCECs stimulated by A.fumigatus conidia for 12h、18h、24h、30h and 36 h.2.HCECs were treated with GA at different concentrations(1.5ng/ml、2.5ng/ml、3.5ng/ml、5ng/ml)for 24 hours,and the toxicity of GA to HCECs was detected using the CCK-8 kit.HCECs were pretreated with different concentrations of GA for 2hours,and then stimulated with A.fumigatus conidia for 16 h.Real-time PCR was used to detect the m RNA expression of CXCL10 in HCECs to select the optimal concentrations of GA.HCECs were pretreated with optimal concentrations of GA for 2 hours,and then stimulated with A.fumigatus conidia for 16 h.Real-time PCR was used to detect the expression of pro-inflammatory cytokine IL-6、IL-23 and anti-inflammatory cytokine IL-10、TGF-β m RNA in HCECs after CXCL10 inhibition.ELISA was used to detect the protein secretion of pro-inflammatory cytokine IL-6、IL-23 and anti-inflammatory cytokine IL-10、TGF-β after 24 h.3.Murine model of A.fumigatus conidia infection at 1d、3d and 5d was established by intratumoral injection.Real-time PCR was used to detect the expression of CXCL10 m RNA in the normal murine corneas and corneas stimulated by A.fumigatus conidia.ELISA was used to detect quantitatively the protein secretion of CXCL10.4.Murine corneas were infected with A.fumigatus conidia after pretreatment of GA,and a slit lamp microscope was used to observe the corneal infectionand label corneal clinical scores at 1d、3d and 5d.Myeloperoxidase(MPO)was used to evaluate neutrophil activity.Hematoxylin-Eosin(HE)staining was used to observe pathological changes in corneal tissue.The healthy C57BL/6 mice were randomly divided into four group: the solvent control group,the A.F group,the GA group,the GA+A.F group,.The infected corneas were collected at 3d for Real-time PCR and ELISA.Real-time PCR was used to detect the expression of proinflammatory cytokine IL-6、IL-23、IL-12、IFN-γ and anti-inflammatory cytokine IL-10、TGF-β.ELISA was used to detect the protein secretion of pro-inflammatory cytokine IL-6、IL-23、IL-12、IFN-γ and anti-inflammatory cytokine IL-10、TGF-β.Results: 1.A.fumigatus conidia increased the CXCL10 m RNA expression and protein secretion in HCECs.m RNA expression peaked at 16 h,protein secretion peaked at 24 h.2.CCK-8 assay showed the concentration of GA less than and eaqual to 5ng/ml can be applied as the effective dose,and 2.5 ng/ml GA has a significant inhibitory effect on the expression of CXCL10 m RNA in HCECs.Therefore,2.5ng/ml GA was used in the following experiments.In HCECs,Real-time PCR illustrated that IL-10,TGF-β,IL-6 and IL-23 increased in A.fumigatus conidia infection group in comparison with the control group.After pretreatment with GA,anti-inflammatory cytokines IL-10、TGF-β were significantly increased while pro-inflammatory cytokine IL-6、IL-23 were significantly decreased in comparison with the A.fumigatus conidia infection group.Further studies by ELISA also showed the change trend of associated inflammatory cytokine at protein levels.3.A.fumigatus conidia infection increased the CXCL10 m RNA expression in the corneal epithelium at 1 day and began to decline at 3 days.The results of ELISA illustrated that CXCL10 protein began to elevate in the corneal epithelium at 1 days,continued to increase at 3 days and decline at 5 days.4.In the murine models of fungal keratitis,the disease scores indicated that infection relieved in the murine models pretreated with GA in comparision with solvent control group at 1d,3d and 5d.The MPO test illustrate the inhibition effect on neutrophil activity.The results of HE showed that a significant decrease in inflammatory cell infiltration in mice with GA pretreated after corneal infection at 3 days.After pretreatment with GA,the Real-time PCR illustrated anti-inflammatory cytokine IL-10、TGF-β were significantly increased while the pro-inflammatory cytokine IL-6、IL-23 were significantly decreased in comparison with the AF group at 3days.Further studies by ELISA also showed the change trend of associated inflammatory cytokine at protein levels.Conclusion: 1.CXCL10 participates in the inflammatory response of Aspergillus fumigatus keratutis.2.CXCL10 could promote the infiltration of inflammatory cells in the infected corneal and up-regulate the expression of inflammatory cytokine.3.Regulation of CXCL10 plays an important role in the inflammation response induced by A.fumigates and might become a new target for clinical diagnosis and treatment.