The Role Of Autophagy In The Innate Immune Response To Fungal Keratitis

Purpose.To determine the role of autophagy in fungal keratitis(FK)caused by A.fumigatus infection.Method.1.Corneal tissue samples were obtained from patients with FK as well as healthy donors,and visualized via transmission electron microscopy(TEM).2.Mice corneas were infected with A.fumigatus.Autophagy-related proteins LC3B-II,Beclin-1,LAMP-1,and p62 in normal,sham-operation and A.fumigatus-infected corneas of C57BL/6 mice at 12 hours,1day and 3 days were tested by Western blot.Normal and infeced mice corneas were visualized via TEM.3.After treatment with autophagy inhibitors 3-MA,CQ,or autophagy inducer rapamycin,autophagy-related proteins were detected by Western blot.Corneas were photographed with slit lamp microscopy and pathological changes were observed by H&E staining.For polymorphonuclear neutrophilic leukocyte(PMN)migration,CXCL-1 was detected by RT-PCR and PMNs were assessed by immunofluorescent staining and observed under TEM.RT-PCR and Western blot were used to test the levels of IL-1?,HMGB1,IL-18,TNF-?,and IL-10.Results.1.The accumulation of autophagosomes in corneas of patients with FK was observed with TEM.2.The expression of LC3B-II,Beclin-1,and LAMP-1 was gradually elevated in corneas after fungal infection at 12 hours,1 day and 3 days,while p62 was down-regulated.The accumulation of autophagosomes in corneas of mice with FK was observed with TEM.3.Treatment with 3-MA or CQ aggravated disease severity and pathological changes compared with infected control group,whereas rapamycin alleviated corneal disease response.4.The expression of LC3B-II,Beclin-1,and LAMP-1 was down-regulated and the produciton of p62 was increased in corneas of mice after 3-MA treatment compared with DMSO control group.Treatment with CQ increased the levels of LC3B-II,Beclin-1,LAMP-1 and p62 compared with PBS control goup.Higher expression of proteins LC3B-II,Beclin-1 and LAMP-1 and lower expression of p62 were detected in corneas after rapamycin treatment versus vehicle control group.5.Treatment with 3-MA and CQ elevated the m RNA level of CXCL-1 and the migration of PMNs compared with infected control group,while rapamycin reduced the expression of CXCL-1 and migration of PMNs.Morphology of PMNs showed less granulation and higher amounts of nuclear euchromatinat in corneas after 3-MA and CQ treatment than in infected control corneas.6.Treatment with 3-MA and CQ enhanced the m RNA and protein levels of IL-1?,HMGB1,IL-18,and TNF-? except IL-10 compared with infected control group,while the expression of IL-1?,HMGB1,IL-18,and TNF-? was down-regulated and the expression of IL-10 was increased in corneas after rapamycin treatment.Conclusion.A.fumigatus infection increases the expression of autophagy in corneas.Inhibition of autophagy aggravates FK caused by A.fumigatus infection,while autophagy inducer alleviates the severity of keratitis.Autophagy plays an anti-inflammatory role in the innate immune response to A.fumigatus keratitis.