Peng Xudong, Zhao Guiqiu, Lin Jing, Qu Jianqiu, Zhang Yingxue, Li Cui
Department of Ophthalmology, The Affiliated Hospital of Qingdao University, NO. 16 Jiangsu Road, Qingdao, 266003, Shandong Province, China.
Department of Biochemistry, Immunology and Microbiology, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI, 48201, USA.
BMC Ophthalmol. 2018 Jul 13;18(1):170. doi: 10.1186/s12886-018-0847-6.
Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. This study aimed to identify the role of PLCγ2 in Dectin-1-mediated Ca Flux and its effect on the expression of proinflammatory mediators at the exposure to Aspergillus fumigatus (A. fumigatus) hyphae antigens in human corneal epithelial cells (HCECs).
The HCECs were preincubated with or without different inhibitors respectively before A. fumigatus hyphae stimulation. Intracellular calcium flux in HCECs and levels of PLCγ2 and spleen-tyrosine kinase (Syk) were detected by fluorescence imaging and Western Blotting. The expression of proinflammatory mediators was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).
We demonstrated that an intracellular Ca flux in HCECs was triggered by A. fumigatus hyphae and could be reduced by pre-treatment with PLCγ2-inhibitor U73122. A. fumigatus hyphae induced PLCγ2 phosphorylation was regulated by Dectin-1 via Syk. Furthermore, PLCγ2-deficient HCECs showed a drastic impairment in the Ca signaling and the secretion of IL-6, CXCL1 and TNF-α.
PLCγ2 plays a critical role for Ca Flux in HCECs stimulated by A. fumigatus hyphae. Syk acts upstream of PLCγ2 in the Dectin-1 signaling pathway. The expressions of proinflammatory mediators induced by A. fumigatus are regulated by the activation of Dectin-1-mediated PLCγ2 signaling pathway in HCECs.
真菌性角膜炎（FK）是一种威胁视力的疾病，因其临床表现复杂、现有治疗方法疗效欠佳以及无法控制的过度先天性炎症而占相当大的比例。磷脂酶C-γ2（PLCγ2）是一种非受体酪氨酸激酶，在先天性免疫早期发挥重要作用。本研究旨在确定PLCγ2在人角膜上皮细胞（HCECs）中Dectin-1介导的钙流中的作用及其对暴露于烟曲霉（A. fumigatus）菌丝抗原时促炎介质表达的影响。
在烟曲霉菌丝刺激之前，分别用不同抑制剂或不用抑制剂对HCECs进行预孵育。通过荧光成像和蛋白质免疫印迹检测HCECs中的细胞内钙流以及PLCγ2和脾酪氨酸激酶（Syk）的水平。通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）测定促炎介质的表达。
我们证明，烟曲霉菌丝可触发HCECs中的细胞内钙流，而用PLCγ2抑制剂U73122预处理可使其减少。烟曲霉菌丝诱导的PLCγ2磷酸化由Dectin-1通过Syk调节。此外，PLCγ2缺陷的HCECs在钙信号传导以及IL-6、CXCL1和TNF-α的分泌方面表现出严重受损。
PLCγ2在烟曲霉菌丝刺激的HCECs中的钙流中起关键作用。Syk在Dectin-1信号通路中位于PLCγ2的上游。烟曲霉诱导的促炎介质的表达受HCECs中Dectin-1介导的PLCγ2信号通路激活的调节。
