Dectin-1 Is Essential For IL-1? Production Through JNK Activation And Apoptosis In Aspergillus Fumigatus Keratitis

Purpose: To investigate the role of phosphorylated JNK in Dectin-1-induced IL-1? production and the role of Dectin-1 in apoptosis in mouse Aspergillus fumigatus(A.fumigatus)keratitis.Methods: 1.Murine model of A.fumigatus corneal infection at 12 h,1d and 2d was established by intratumoral injection.The m RNA and protein expressions of Dectin-1 and IL-1? were detected by PCR and Western Bolt.The localization of Dectin-1 in the normal or infected corneas of mice was detected by immunofluorescence.2.The corneas of mice were pretreated with Dectin-1 si RNA 1 day before infection and at the time of A.fumigatus infection.Slit lamp was used to observe the inflammatory response of mice corneas,and clinical scores were recorded.PCR or Western Bolt was used to detect the expressions of Dectin-1,JNK,p-JNK,and IL-1?.3.The corneas of mice or THP-1 macrophages were pretreated with JNK inhibitor SP600125 before A.fumigatus infection.PCR and Western blot were used to detect the expression of IL-1?.4.The corneas of mice were pretreated with Dectin-1 si RNA 1 day before infection and at the time of A.fumigatus infection.PCR and Western Bolt were used to detect the expressions of Bcl-2,Bax,cytochrome-c(cyt-c),caspase-8,caspase-9 and caspase-3.Results: 1.Compared with uninfected mice corneas,Dectin-1 m RNA and protein level was significantly increased at 12 h,1d and 2d after A.fumigatus infection.IL-1? m RNA and protein level was significantly increased at 12 h,1d and 2d after A.fumigatus infection.2.The corneas of mice were pretreated with Dectin-1 si RNA before A.fumigatus infection.Dectin-1 si RNA treatment reduced disease severity of fungal keratitis compared with the scrambled control.A significant difference was also observed in clinical scores of the Dectin-1 si RNA-treated corneas versus the scrambled control after infection.Dectin-1 si RNA treatment before infection significantly suppressed JNK phosphorylation,IL-1? m RNA and protein expressions compared with infected scrambled control.3.The corneas of mice or THP-1 macrophages were pretreated with JNK inhibitor SP600125 before A.fumigatus infection.In vivo and vitro,pretreatment with SP600125 before infection decreased IL-1? production at m RNA and protein levels compared with infected DMSO control.4.Relative Bcl-2 m RNA level was significantly decreased in Dectin-1 si RNA pretreatment of the A.fumigatus-infected corneas compared with infected scrambled si RNA control;while,the m RNA levels of Bax,cyt-c,caspase-9,and caspase-3 were significantly increased.No significant difference was detected on m RNA level of caspase-8 between infected scrambled si RNA group and infected Scrambled si RNA control.Dectin-1 si RNA treatment significantly suppressed the Bcl-2 protein expression when compared to scrambled si RNA control after infection.On the contrary,protein expressions of Bax,cyt-c,cleaved-caspase-9,and cleaved-caspase-3 were increased compared infected Dectin-1 si RNA pretreatment group with infected Scrambled si RNA control.No significant difference was detected on protein level of cleaved-caspase-8 between infected Scrambled si RNA group and infected scrambled si RNA control.Conclusions: Dectin-1 and IL-1? expressions were increased in mice with A.fumigatus keratitis.The phosphorylation of JNK mediated Dectin-1-induced IL-1? production.Dectin-1 enhanced anti-apoptotic activity in response to A.fumigatus infection,which suppressed apoptosis mediated anti-inflammatory response in mouse A.fumigatus keratitis.Dectin-1 deficiency-induced apoptosis seemed to be executed through intrinsic pathway rather than extrinsic.