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

ABSTRACT

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.

KEYWORDS:

• LOX-1
• neutrophil
• apoptosis
• fungal load
• A. fumigatus keratitis

Author’s contributions

Cui Li, Kun He and Min Yin carried out most of the experimental work. Jing Lin, Nan Jiang and Yawen Niu conducted cellular experiments. Qiang Xu conducted the cell culture and immunofluorescent staining. Qian Wang conducted flow cytometry. Cui Li, Kun He, Min Yin, Qiuqiu Zhang and Guiqiu Zhao supervised the project and wrote the manuscript. All authors reviewed the manuscript.

Disclosure statement

The authors declare no competing financial interests.

Key messages

1. Apoptosis occurred in C57BL/6 mice corneas infected with A. fumigatus

2. LOX-1 inhibition decrease fungal load in A. fumigatus keratitis.

3. LOX-1 inhibition decrease neutrophil apoptosis in A. fumigatus keratitis through Fas and Caspase pathways.

Additional information

Funding

This work was supported by National Natural Science Foundation of China (81800800)National Science Foundation of China [81800800];