The Role And Regulatory Mechanism Of MST4 In Anti-fungal Immune Response Of Corneal Epithelial Cells

Purpose: To investigate the role and molecular mechanism of mammalian Ste20-like kinase 4(MST4)in the immune response of corneal epithelial cells to Aspergillus fumigatus(A.fumigatus).Method: 1.A.fumigatus infected the cornea of C57BL/6 mice to establish a mouse model of fungal keratitis,we observed the clinical score of mice corneas and used Western blot to detect the expression of MST4 protein in normal mice corneas and corneas after A.fumigatus infection for 12 h and 1,3,5 days.The expression of MST4 protein in normal immortalized human corneal epithelial cells(HCECs)and HCECs stimulated by A.fumigatus hyphae for 12 hours and 24 hours was detected by Western blot.Immunofluorescence staining was used to detect the expression and localization of MST4 in normal mice corneas and corneas 3 days after infection.2.HCECs were pretreated with exogenous MST4 recombinant protein(2,5,10 ng/m L)for 2 hours,and then stimulated with A.fumigatus for 12 hours and 24 hours.PCR and ELISA were used to detect IL-1?,IL-6 and IL-8 m RNA and protein expression in each group of HCECs.HCECs were pretreated with curdlan or Laminarin for 10 hours,followed by stimulated with A.fumigatus for 24 hours.The expression of MST4 protein in HCECs was detected by Western blot.4.HCECs were pretreated with exogenous MST4 recombinant protein(2,5,10 ng/m L)for 2 hours,and then added Curdlan to stimulate HCECs for 10 hours and 24 hours.PCR and ELISA were used to detect m RNA and protein expressions of IL-1?,IL-6 and IL-8 in each group of HCECs.RAW 264.7 mouse macrophages were pretreated with exogenous MST4 recombinant protein(2,5 ng/m L)for 2 hours,and then RAW 264.7 cells were stimulated with Curdlan for 8 hours.The expression of IL-1? and IL-6 in each group of RAW 264.7 cells was detected by PCR.5.HCECs were treated with exogenous MST4 recombinant protein(2,5,10,50 ng/m L)for 24 hours,48 hours and 72 hours,respectively,and the proliferation ability of HCECs was detected by CCK8 kit and cell count.Results: 1.The expressions of MST4 protein in mice corneas were significantly higher than that in normal cornea at 1 day,3 days and 5 days after infection with A.fumigatus.The expression of MST4 protein reached a peak at 3 days after infection.The expressions of MST4 protein in HCECs stimulated with A.fumigatus for 12 hours and 24 hours was significantly higher than normal group.The fluorescence staining of MST4 was significantly enhanced in the mouse cornea at 3 days after A.fumigatus infection and MST4 mainly located in the corneal epithelium.2.A.fumigatus significantly promoted the expression of proinflammatory cytokines(IL-1?,IL-6,IL-8)m RNA and protein in HCECs.Compared with the A.fumigatus infected group,the m RNA and protein levels of above cytokines(IL-1?,IL-6,IL-8)of HCECs were significantly down-regulated by recombinant MST4(2,5,10 ng/ml)before the A.fumigatus.Compared with the A.fumigatus infected group,pretreatment with Curdlan prior to A.fumigatus further up-regulated the protein level of MST4,while pretreatment with Laminarin significantly reduced the protein expression of MST4.4.Curdlan significantly promoted the m RNA and protein expression of pro-inflammatory cytokines(IL-1?,IL-6,IL-8).The m RNA and protein levels of the above cytokines(IL-1?,IL-6,IL-8)were significantly down-regulated with recombinant MST4(2,5,10 ng/ml)before Curdlan stimulation compared with the group treated with Curdlan alone.In addition,Curdlan significantly promoted the expression of proinflammatory cytokines(IL-1?,IL-6)m RNA in RAW264.7 cells.Compared with the Curdlan-treated control group,cytokine(IL-1?,IL-6)m RNA level was significantly downregulated with recombinant human MST4(2,5 ng/ml)before Curdlan stimulation.5.A.fumigatus or Curdlan significantly promoted the phosphorylation of Syk in HCECs,while MST4 inhibited the level of Syk phosphorylation induced by A.fumigatus or Curdlan.6.There was no significant difference in proliferation ability between HCECs treated with different concentrations of MST4 recombinant protein(2,5,10,50 ng/m L)for 24 hours,while MST4 recombinant protein administration for 48 and 72 hours promoted HCECs proliferation.The proliferative capacity of HCECs treated with MST4 for 48 hours increased in an MST4 dose-dependent manner.Conclusion: A.fumigatus stimulates the C57BL/6 mice corneas and HCECs to produce MST4 which could be regulated by Dectin-1.Recombinant MST4 can inhibit A.fumigatus and Dectin-1 mediated inflammatory responses by inhibiting Syk phosphorylation in HCECs.MST4 also promotes HCECs proliferation.