抗菌肽人β-防御素9（HBD9）的体外研究：信号通路与病原体相关反应（美国眼科学会论文）
In vitro studies on the antimicrobial peptide human beta-defensin 9 (HBD9): signalling pathways and pathogen-related response (an American Ophthalmological Society thesis).
作者信息
Dua Harminder S, Otri Ahmad Muneer, Hopkinson Andrew, Mohammed Imran
机构信息
Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, England.
出版信息
Trans Am Ophthalmol Soc. 2014 Jul;112:50-73.
PURPOSE
Human β-defensins (HBDs) are an important part of the innate immune host defense at the ocular surface. Unlike other defensins, expression of HBD9 at the ocular surface is reduced during microbial infection, but activation of toll-like receptor 2 (TLR2) in corneal epithelial cells has been shown to up-regulate HBD9. Our purpose was to test the hypothesis that TLR2 has a key role in the signalling pathway(s) involved in the overexpression or underexpression of HBD9, and accordingly, different pathogens would induce a different expression pattern of HBD9.
METHODS
The in vitro RNAi silencing method and response to dexamethasone were used to determine key molecules involved in signalling pathways of HBD9 in immortalized human corneal epithelial cells. The techniques included cell culture with exposure to specific transcription factor inhibitors and bacteria, RNA extraction and cDNA synthesis, quantitative real-time polymerase chain reaction, and immunohistology.
RESULTS
This study demonstrates that TLR2 induces HBD9 mRNA and protein expression in a time- and dose-dependent manner. Transforming growth factor-β-activated kinase 1 (TAK1) plays a central role in HBD9 induction by TLR2, and transcription factors c-JUN and activating transcription factor 2 are also involved. Dexamethasone reduces TLR2-mediated up-regulation of HBD9 mRNA and protein levels in mitogen-activated protein kinase phosphatase 1 (MKP1)-dependent and c-JUN-independent manner. HBD9 expression differs with gram-negative and gram-positive bacteria.
CONCLUSIONS
TLR2-mediated MKPs and nuclear factor-κB signalling pathways are involved in HBD9 expression. TAK-1 is a key molecule. These molecules can be potentially targeted to modulate HBD9 expression. Differential expression of HBD9 with different bacteria could be related to differences in pathogen-associated molecular patterns of these organisms.
目的
人β-防御素（HBDs）是眼表先天性免疫宿主防御的重要组成部分。与其他防御素不同，在微生物感染期间，眼表HBD9的表达会降低，但已表明角膜上皮细胞中Toll样受体2（TLR2）的激活会上调HBD9。我们的目的是检验以下假设：TLR2在参与HBD9过表达或低表达的信号通路中起关键作用，因此，不同病原体将诱导不同的HBD9表达模式。
方法
采用体外RNA干扰沉默方法和对地塞米松的反应，以确定永生化人角膜上皮细胞中HBD9信号通路中的关键分子。技术包括用特定转录因子抑制剂和细菌进行细胞培养、RNA提取和cDNA合成、定量实时聚合酶链反应以及免疫组织学。
结果
本研究表明，TLR2以时间和剂量依赖性方式诱导HBD9 mRNA和蛋白表达。转化生长因子-β激活激酶1（TAK1）在TLR2诱导HBD9过程中起核心作用，转录因子c-JUN和激活转录因子2也参与其中。地塞米松以丝裂原活化蛋白激酶磷酸酶1（MKP1）依赖性和c-JUN非依赖性方式降低TLR2介导的HBD9 mRNA和蛋白水平上调。HBD9的表达因革兰氏阴性菌和革兰氏阳性菌而异。
结论
TLR2介导的MKPs和核因子-κB信号通路参与HBD9表达。TAK-1是关键分子。这些分子可能是调节HBD9表达的潜在靶点。HBD9在不同细菌中的差异表达可能与这些生物体病原体相关分子模式的差异有关。
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