The Research Of Candidalysin Regulates The Immune Inflammatory Response To Candida Albicans Keratitis Through TREM-1

Objective: To investigate the molecular mechanism of Candidalysin regulates the immune inflammatory response to Candida albicans keratitis through TREM-1.Methods: 1.The corneas of mice were infected with Candida albicans to establish a mouse model of Candida albicans keratitis at 1d,3d and 5d.The expression level of TREM-1m RNA in mouse cornea were detected by PCR method.The expression level of TREM-1protein in mouse cornea were detected by Western-blot method.2.Eyes of mice were pretreated with different concentrations of TREM-1 inhibitor GF9(0.1,0.2 and 0.5 mg/ml)before infection with Candida albicans.The expression of TREM-1 m RNA in the corneas of mice were detected by PCR,and the GF9 concentration with the best inhibitory effect was screened,and in the next experiments the corneas of mice were pretreated with the optimal concentration of GF9.The expression of TREM-1 protein in the corneas of normal mice,Candida albicans infected mice and the GF9(0.2 mg/ml)pretreated mice were detected by Western blot.The expression and localization of TREM-1 protein in the corneas of mice in each group were detected by immunofluorescence staining.The expression of inflammatory cytokines(TNF-α,IL-1β,IL-6)m RNA in the corneas of mice in each group were detected by PCR.3.The morphology and inflammation degree of mice corneas were observed after 1,3 and 5 days of Candida albicans infection.The clinical scores of mice corneas were recorded.The relation between clinical scores of mice models and the expression of TREM-1 m RNA after Candida albicans infection was analyzed by Pearson correlation analysis.4.Eyes of mice were pretreated with GF9 for 24 h or stimulated with Candidalysin for 2h before infection with Candida albicans.The experimental groups were normal group,Candida albicans infected group,GF9 pretreated group,Candidalysin pretreated group and GF9+Candidalysin pretreated group.Detected the expression of TREM-1,TNF-α,IL-1β and IL-6 m RNA in the corneas of mice in each group by PCR.Detected the expression of TREM-1,ERK and p-ERK protein in the corneas of mice in each group by Western blot.5.Eyes of mice were pretreated with ERK pathway inhibitor PD98059 for 24 h or stimulated with Candidalysin for 2h before infection with Candida albicans.Detected the expression of inflammatory cytokines(TNF-α,IL-1β,IL-6)m RNA in the corneas of mice in each group by PCR.Detected the expression of ERK and p-ERK protein in the corneas of mice in each group by Western blot.Results: 1.The expression of TREM-1 m RNA in the cornea of mice infected with Candida albicans for 1 day,3 days and 5 days was significantly higher than that of the normal group.The level of TREM-1 m RNA reached its peak after 3 days infection.The expression of TREM-1 protein in the cornea of mice infected with C.albicans for 3 days and 5 days was significantly higher than that of normal cornea.The level of TREM-1 protein decreased on day 5 of infection compared to day 3.2.Compared with the group infected with Candida albicans alone,different concentrations of GF9 pretreatment can significantly reduce the expression of TREM-1 m RNA in the corneas of mice infected with Candida albicans.The best inhibition effect was achieved when the GF9 concentration was 0.2 mg/ml.Pretreated mice corneas with GF9(0.2mg/ml)can significantly reduce the protein level of TREM-1and the m RNA level of inflammatory cytokines(TNF-α,IL-1β,IL-6)in mice corneas.3.The clinical scores of mice corneas infected with Candida albicans for 1,3,5 days were higher than that of normal mice corneas.And the clinical scores of mice corneas reached its peak after 3 days infection.Pearson correlation analysis showed significant relation between clinical scores of mice model and the expression of TREM-1 m RNA after Candida albicans infection.4.Compared with the group infected with Candida albicans alone,the expression of TREM-1 m RNA and protein in corneas of mice pretreated with Candidalysin were significantly increased,and the expression of inflammatory cytokines(TNF-α,IL-1β,IL-6)m RNA in the corneas of mice pretreated with Candidalysin were also significantly increased.Compared with the Candidalyisn pretreatment group,the expression of TREM-1 m RNA and protein in the corneas of mice pretreated with GF9+Candldalysin were significantly decreased,and the expression of inflammatory cytokines(TNF-α,IL-1β,IL-6)m RNA in the corneas of mice pretreated with GF9+Candldalysin were also significantly decreased.5.Compared with the group infected with Candida albicans alone,Candidalysin pretreatment increased the ERK phosphorylation level in the corneas of mice infected with Candida albicans,while GF9 pretreatment inhibited the level of ERK phosphorylation induced by Candida albicans or Candidalysin.6.Compared with the Candidalysin pretreatment group,pretreated mice corneas with ERK inhibitor PD98059 before Candidalysin treatment can significantly reduce the expression of inflammatory cytokines(TNF-α,IL-1β,IL-6)m RNA in mice corneas,the level of ERK phosphorylation in mice corneas were also decreased.Conclusion: Candida albicans infection can increase the expression of TREM-1 in corneas of mice.Candidalysin can enhance the inflammatory response to Candida albicans keratitis through the TREM-1 and regulate cytokine expression by enhancing the phosphorylation of the downstream signaling kinase ERK.