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.

中文翻译：

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LOX-1 调节烟曲霉角膜炎中的中性粒细胞凋亡和真菌负荷

摘要

目的

确定 LOX-1 是否调节烟曲霉角膜炎中的中性粒细胞凋亡和真菌负荷。

方法

Fas、FasL、CASP3、CASP8、CASP9 和 BCL2 在 C57BL/6 小鼠的正常和感染角膜中进行了测试。在有或没有 LOX-1 中和抗体或抑制剂 (Poly I) 预处理的情况下，用烟曲霉感染小鼠角膜。记录临床评分并测试HE染色。通过平板计数观察小鼠角膜中的真菌负荷。在有或没有 LOX-1 中和抗体或 Poly I 预处理的情况下，用 75% 乙醇杀死的烟曲霉刺激多形核中性粒细胞 (PMN) 。PCR、蛋白质印迹和免疫染色测试 Fas、FasL、CASP3、CASP8、CASP9 的表达, BCL2 和裂解的 caspase-3。通过免疫荧光染色评估 PMN 浸润和 TUNEL 阳性细胞。流式细胞仪检测凋亡中性粒细胞的百分比。

结果

感染烟曲霉的 C57BL/6 小鼠角膜中 Fas、Fas 配体、caspase-8、caspase-9 和 caspase-3 mRNA 水平显着升高比正常的角膜。Poly I 治疗在感染后 (pi) 后 3、5 和 7 天减轻了严重程度并降低了临床评分。HE染色显示LOX-1抑制后角膜组织浸润较少。平板计数实验表明，Poly I治疗组角膜中活菌数明显少于对照组。LOX-1 中和抗体或 Poly I 处理显着降低中性粒细胞浸润、TUNEL 阳性细胞数量、Fas、Fas 配体、caspase-8、caspase-9、caspase-3、cleaved caspase-3 的表达和百分比与对照角膜相比，凋亡中性粒细胞。LOX-1 中和抗体治疗显着降低了中性粒细胞中的 Fas、FasL、CASP3、CASP8、CASP9 和切割的 caspase-3 表达。

结论

LOX-1 抑制降低烟曲霉角膜炎中的中性粒细胞凋亡和真菌负荷。