LOX-1 Regulates Neutrophil Apoptosis and Fungal Load in A. Fumigatus Keratitis

Purpose: To determine whether LOX-1 regulates neutrophil apoptosis and fungal load in A. fumigatus keratitis.

Methods: Fas, FasL, CASP3, CASP8, CASP9 and BCL2 were tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or inhibitor (Poly I). Clinical score was recored and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Poly morphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or Poly I. PCR, western blot and immunostaining tested expression of Fas, FasL, CASP3, CASP8, CASP9, BCL2 and cleaved caspase-3. PMNs infiltration and TUNEL-positive cells were assessed by immunofluorescent staining. Flow cytometry assay tested the percentage of apoptosis neutrophils.

Results: Fas, Fas ligand, caspase-8, caspase-9 and caspase-3 mRNA levels were significantly higher in C57BL/6 mice corneas infected with A. fumigatus than normal corneas. Poly I treatment alleviated the severity and decreased clinical score at 3, 5 and 7 days post infecrion (p.i.). HE staining showed less infiltration in corneal tissue after LOX-1 inhibition. Plate counting experiment showed that number of viable fungus in corneas of Poly I treated group was significantly less than control group. LOX-1 neutralizing antibody or Poly I treatment significantly decreased neutrophil infiltration, the quantity of TUNEL-positive cells, the expression of Fas, Fas ligand, caspase-8, caspase-9, caspase-3, cleaved caspase-3 and the percentage of apoptosis neutrophils compared with control corneas. LOX-1 neutralizing antibody treatment significantly decreased Fas, FasL, CASP3, CASP8, CASP9 and cleaved caspase-3 expression in neutrophils.

Conclusion: LOX-1 inhibition decrease neutrophil apoptosis and fungal load in A. fumigatus keratitis.