Mincle In The Innate Immune Response Of Mice Fungal Keratitis

Aim: To investigate the role of macrophage inducible C-type lectin(Mincle)in mice fungal keratitis induced by A.fumigatus.To work out how Mincle participates in the innate immune response of mice fungal keratitis by influencing the expression of cytokines and chemokines,as well as the generation of nitric oxide(NO)in mice cornea tissues.Methods: C57BL/6 mice corneas were infected with active A.fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody(mincle Ab),taking DMSO or Ig G as control group respectively.The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score.The m RNA and protein expression of Mincle were assessed using real time PCR and immunostaining.The m RNA and protein expression of cytokines(IL-1?,TNF-? and IL-6)were determined by real time PCR and ELISA.The m RNA expression of chemokines(CXCL-1 and MIP-2)in mice corneas were detected by real time PCR.Neutrophil infiltration was observed by immunostaining.The levels of nitric oxide(NO)generated by corneas were tested by Griess reaction.Results: 1.Mincle m RNA and protein levels were higher in infected corneas than normal corneas of C57BL/6 mice,the m RNA level peaked on 1 day post infection.The expression of Mincle was significantly increased after pretreatment with Mincle agonist TDB,while the fluorescence intensity of Mincle was significantly decreased after pretreatment with Mincle Ab,indicating that Mincle expression was effectively blocked.2.When infected by A.fumigatus without other treatment,the mice corneas were mild turbidity with irregular ulcers.There were no significant differences between corneas in DMSO / Ig G control group and AF group,no statistical difference of clinical score was observed.Compared with the DMSO control group,the A.fumigatus keratitis of mice in TDB pretreatment group showed smaller ulcer area and less depth.The difference of clinical score was statistically significant(P<0.01).However,the corneas from Mincle Ab pretreatment group suffered with larger corneal ulcer area,deeper invasion and increased probability of corneal perforation compared with the control group.The difference of clinical score was statistically significant(P<0.01).3.When pretreated with Mincle agonist TDB,the m RNA and protein levels of IL-1?,TNF-? and IL-6 in infected corneas were significantly increased compared with the DMSO control group(P<0.01).When pretreated with Mincle Ab,the m RNA and protein levels of IL-1?,TNF-? and IL-6 in infected corneas were significantly decreased compared with the Ig G control group(P<0.01).4.The expression of CXCL1 and MIP-2 m RNA levels were up-regulated in TDB pretreatment group and down-regulated in Mincle Ab pretreatment group(P<0.01),coincide with neutrophil aggregation degree in corneas showed by immunostaining.Neutrophils can be hardly discovered in normal cornea tissues.In AF group and DMSO/Ig G pretreatment group,neutrophils can be observed in the cornea tissue.There is no significant differences between AF group and control group.In the TDB pretreated group we observed a great deal of neutrophils gathered around the lesion site,and in the Mincle Ab group,aggregation of neutrophils was inhibited.5.Nitric oxide can be detected in normal mice corneas.It was promoted in TDB pretreatment group compared with DMSO control group,and decreased in mincle Ab pretreatment group compared with Ig G control group.Conclusion: Mincle is involved in the innate immune response of A.fumigatus keratitis in mice.It can promote inflammatory response by regulating secretion of several cytokines and chemokines,collecting neutrophils to lesion sites,and by regulating the concentration of NO in cornea tissue,so as to play a cytotoxic role in killing fungi directly.