Mincle Regulates Neutrophils Apoptosis In A.fumigatus Keratitis

Objective: To determine whether macrophage inducible C-type lectin(Mincle)regulates neutrophils apoptosis in A.fumigatus keratitis.Methods: Mincle was tested in normal and infected cornea of C57BL/6.The infectious corneal was observed under the microscope of 12 hours,1 day,3 days and 5 days after stimulation,and then the whole cornea was collected.Mice were pre-treated with TDB and mincle neutralizing antibody before mice were infected.HE staining was used to test disease response.PCR,Western blot,immunostaining,and TUNEL staining assay was also used to test the expression of Mincle,inflammatory mediators and PMNs infiltrate.Neutrophils were pretreated with Mincle neutralizing antibody and TDB,and then were stimulated with 75% ethanol-killed A.Fumigatus.PCR,Western blot and flow cytometry was used to test inflammatory mediators and neutrophils and macrophages apoptosis.Neutrophils and macrophages were extracted from C57BL/6 mice,and Mincle was tested in normal and stimulated neutrophils and macrophages.Cells pretreated with Mincle neutralizing antibody or Ig G;Mincle agonist TDB or PBS,and then stimulated with A.fumigatus.The m RNA level of Fas,Fas L and Caspase3 were evaluated by PCR.Neutrophils apoptosis were also assessed by flow cytometry.Results: Mincle m RNA and protein were higher in C57BL/6 mice infected cornea than normal control,saving clinical score and HE assay revealed differences.After treatment with Mincle neutralizing antibody,Fas,Fas L and Caspase3 m RNA levels,neutrophils infiltration and TUNEL-positive cells in C57BL/6 corneas were significantly increased compared with treatment with control Ig G.In contrast,mice treated with TDB showed decreased Fas,Fas L and Caspase3 m RNA levels,neutrophils infiltration and TUNEL-positive cells.Mincle m RNA levels were higher in normal and stimulated neutrophils and macrophages.The m RNA levels of Fas,Fas L and Caspase3 were significantly increased in Mincle neutralizing antibody treatment compared with Ig G control in neutrophils,correlated with significantly more neutrophils apoptosis,while comparing TDB with normal control,Fas,Fas L and Caspase3 m RNA were significantly decreased and less neutrophils or macrophages apoptosis.Conclusions: These data provided evidence that Mincle expression was higher in infected cornea compared with normal control.TDB treatment decreased apoptotic gene levels,caspase-3 activation,neutrophils infiltration,TUNEL-positive cells and neutrophils apoptosis compared with normal control,which were increased in Mincle neutralizing antibody treatment compared with Ig G control.Mincle also inhibits neutrophil apoptosis by inhibiting the binding of FAS and FASL to decrease the expression of Caspase3.