Investigation On The Effect And Mechanism Of Cathelicidin During Aspergillus Fumigatus Keratitis

Objective:To investigate the effect of Cathelicidin on phagocytosis or autophagy of neutrophils during Aspergillus fumigatus(A.fumigatus)keratitis.Methods:1.To establish mice models with A.fumigatus keratitis,RT-PCR and Western blot were used to detect the expression of Cathelicidin(CRAMP)m RNA and protein in mice corneas at 0,1,2,and 4 days post infection.The expression and localization of Cathelicidin(LL-37)in humans corneas from patients with A.fumigatus keratitis and healthy donors were determined by RT-PCR,Western blot,and immunofluorescence.2.The corneas of mice were interfered with exogenous CRAMP or phosphate buffer(PBS).Slit lamp photography and clinical score assessed the efficacy of CRAMP.RT-PCR and ELISA assays detected the expression of pro-inflammatory factors in mice corneas.And hematoxylin-eosin(HE)staining assessed the corneal tissue damage.The amount and proportions of neutrophil recruitment were analyzed by flow cytometry.Periodate-Schiff staining(PAS)and Colony forming unit(CFU)counting assays measured fungal loads in mice corneas.3.In vitro,mice neutrophils pretreated with CRAMP or PBS were co-cultured with carboxyfluorescein succinyl amino ester(CFSE)labeled A.fumigatus conidia.Flow cytometry analyzed the effect of CRAMP on neutrophils’ phagocytosis.Injecting conidia into the abdominal cavity of mice,the impact of CRAMP on neutrophils’ phagocytosis in vivo was analyzed by flow cytometry.4.Human neutrophils were treated with or without CXCR2 antagonist SB225002 or phospholipase Cγ(PLCγ)inhibitor U73122 before LL-37 or PBS treatment,which were finally co-cultured with CFSE-labeled conidia.The changes in phagocytosis levels in each group were evaluated by flow cytometry.The phosphorylation levels of PLCγ in each group were detected by Western blot.5.In vitro,mice neutrophils pretreated with CRAMP or PBS were co-cultured with CFSE-labeled conidia.Neutrophils were then washed and cultured for an indicated time.Flow cytometry analyzed the effect of CRAMP on neutrophils’ ability to clear intracellular conidia in vitro.In vivo,CFSE-labeled conidia were injected into the peritoneal cavity of mice.Flow cytometry was used to analyze the effect of CRAMP on neutrophils’ scavenging ability of intracellular conidia in vivo.6.Human neutrophils were pretreated with or without SB225002 or the autophagy inhibitor bafilomycin A before LL-37 or PBS treatment.Finally,they were co-cultured with CFSE-labeled conidia,and the changes in intracellular fungicidal ability in each group were evaluated by flow cytometry.RT-PCR and Western blot detected each group’s expressions of autophagy-related genes and proteins.Results:1.Increased expression of LL-37 or CRAMP in the cornea of humans or mice infected with A.fumigatus.2.After 2 days of CRAMP treatment,the lesion degree of A.fumigatus keratitis in mice was reduced.After 4 days of treatment,the clinical scores of mice cornea treated with CRAMP were significantly lowered.In line with the changing trend of lesion degree,CRAMP decreased the expression of pro-inflammatory cytokines IL-6 、TNF-a 、IL-1β and the levels of neutrophil infiltration in corneal tissue compared with the PBS-treated group.PAS staining and CFU counting assays showed that the number of residual A.fumigatus in CRAMP-treated corneas was significantly lower than that in the PBS-treated group.3.Flow cytometry showed that CRAMP promoted neutrophils to phagocytize conidia in vivo and in vitro.4.LL-37 enhances the phagocytosis of neutrophils by promoting intracellular PLCγ phosphorylation through its functional receptor CXCR2 on neutrophils..5.Flow cytometry showed that CRAMP enhanced the intracellular fungal clearance of neutrophils in vivo and in vitro.6.LL-37/CXCR2 up-regulated the expression of autophagy related proteins(Beclin-1 and LC3-II)to accelerate the removal of intracellular conidia.Conclusion:Cathelicidin inhibits the inflammatory response and reduces fungal loads during A.fumigatus keratitis.Cathelicidin can accelerate neutrophils to phagocytose and degrade A.fumigatus conidia.Mechanistically,LL37/CXCR2 activates PLC γ in neutrophils to enhance phagocytosis and promotes neutrophils’ autophagy to eliminate intracellular conidia.