Department of Ophthalmology, the Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Basic Medical Sciences, Qingdao University, Qingdao, China.
Immunol Cell Biol. 2018 Apr;96(4):401-412. doi: 10.1111/imcb.12010. Epub 2018 Feb 26.
Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. A phosphorylated glycoprotein secreted by immunocompetent cells, osteopontin (OPN) mediates cluster formation of the host fungal receptors and enhances the phagocytosis and clearance of pathogenic fungi. However, whether OPN production and function occurs in fungal keratitis is unknown. OPN expression in Aspergillus fumigatus keratitis patient corneas was assessed by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. Human neutrophils, THP-1 macrophages and corneal epithelial cells (HCECs) stimulated with A. fumigatus were utilized for in vitro experiments. Mouse models of A. fumigatus keratitis were developed by intrastromal injection for in vivo experiments. Using siRNAs, neutralizing antibodies, recombinant proteins and inhibitors, the production and role of OPN in A. fumigatus infection was assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blotting and bioluminescence image acquisition. We observed increased corneal OPN expression in A. fumigatus keratitis patients and mouse models compared to controls. OPN production in response to A. fumigatus infection was dependent on LOX-1 and Erk1/2. Compared to controls, OPN knockdown impaired proinflammatory cytokine IL-1β production, which was dependent on 4E-BP1. OPN knockdown decreased myeloperoxidase levels, and resulted in decreased neutrophil recruitment, higher fungal load and increased apoptosis in mouse A. fumigatus keratitis. Our results indicate that OPN is a critical component of the antifungal immune response and is essential for effective neutrophil recruitment, inflammatory cytokine production and apoptosis in A. fumigatus keratitis.
真菌性角膜炎是角膜溃疡的主要原因，导致严重的视力损害和失明。免疫活性细胞分泌的磷酸化糖蛋白骨桥蛋白（OPN）介导宿主真菌受体的簇形成，并增强对致病性真菌的吞噬和清除。然而，真菌性角膜炎中是否存在 OPN 的产生和功能尚不清楚。通过定量聚合酶链反应（qRT-PCR）和免疫荧光评估曲霉菌角膜炎患者角膜中 OPN 的表达。利用曲霉菌刺激的人中性粒细胞、THP-1 巨噬细胞和角膜上皮细胞（HCECs）进行体外实验。通过基质内注射建立烟曲霉菌角膜炎小鼠模型进行体内实验。通过使用 siRNA、中和抗体、重组蛋白和抑制剂，通过临床评估、qRT-PCR、免疫荧光、western blot 和生物发光图像采集评估 OPN 在烟曲霉菌感染中的产生和作用。与对照组相比，我们观察到曲霉菌角膜炎患者和小鼠模型中角膜 OPN 表达增加。对烟曲霉菌感染的 OPN 产生依赖于 LOX-1 和 Erk1/2。与对照组相比，OPN 敲低可损害促炎细胞因子 IL-1β 的产生，这依赖于 4E-BP1。OPN 敲低降低髓过氧化物酶水平，导致小鼠烟曲霉菌角膜炎中中性粒细胞募集减少、真菌负荷增加和凋亡增加。我们的研究结果表明，OPN 是抗真菌免疫反应的关键组成部分，对于烟曲霉菌角膜炎中有效的中性粒细胞募集、炎症细胞因子产生和凋亡是必需的。
