Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients
Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients
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活性氧激活的 NLRP3 炎症小体在高渗透压应激的人角膜上皮细胞和环境诱发的干眼患者中引发炎症
DOI：
10.1016/j.exer.2015.02.013
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发表时间：
2015-05-01
影响因子：
3.4
通讯作者：
Chen, Wei
中科院分区：
文献类型：
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作者：
Zheng, Qinxiang;Ren, Yueping;Chen, Wei
在干眼 (DE) 疾病的研究中，已发现泪膜高渗透压与受体诱导的促炎细胞因子和趋化因子释放增加引起的炎症严重程度之间存在关联。这些免疫反应可能是由炎症小体介导的，炎症小体是安装在 NLRP3 蛋白周围的大分子复合物，并且可以通过活性氧 (ROS) 的过度生成来激活。因此，在本研究中，我们确定是否： a) ROS 激活的 NLRP3 炎症小体介导人角膜上皮细胞 (HCEC) 中高渗应激诱导的炎症； b) ROS-NLRP3-IL-1 β轴激活与环境诱导的DE相关。在存在和不存在 ROS 抑制剂 N-乙酰基-L-半胱氨酸 (NAC) 的情况下，将永生化 HCEC 暴露于 500 mOsm 培养基。用 NLRP3 siRNA 或阴性对照 (NC) siRNA 转染的 HCEC。使用探针 2',7'-二氯荧光素二乙酸酯 (DCFH-DA) 通过荧光分析测量细胞内 ROS。实时 PCR 评估了 NLRP3、ASC、pro-caspase-1 和 pro-IL-1 beta mRNA 水平。蛋白质印迹分析评估 NLRP3 蛋白表达，而 caspase-1 活性则通过荧光测定法测定。通过 ELISA 评估生物活性 IL-1 β 释放。还评估了环境诱发的 DE 患者和正常受试者的结膜上皮细胞和泪液样本中 ROS 的产生、NLRP3 炎性体和 pro-IL-1 β 基因表达以及 IL-1 β 分泌。 NAC 抑制高渗透压诱导的 ROS 水平、NLRP3 炎性体形成和激活、caspase-1 活性和 IL-1 β 释放的升高。另一方面，NLRP3 siRNA 敲低抑制高渗应激诱导的 NLRP3 激活，从而导致 ASC、pro-caspase-1 和 pro-IL-1 beta mRNA 下调，随后抑制相关的 caspase-1 活性和 IL-1 beta 分泌。此外，在环境诱发的DE患者的眼表样本中，ROS生成、NLRP3、ASC、pro-caspase-1和pro-IL-1β基因表达以及IL-1β分泌均上调。综上所述，NLRP3 介导由 ROS 生成增加引发的先天免疫反应，诱导高渗应激的 HCEC 发生炎症。 ROS-NLRP3-IL-1 beta 信号通路可能在环境诱导的 DE 发育中发挥启动作用。 (C) 2015 Elsevier Ltd. 保留所有权利。
In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1 beta axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-L-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1 beta mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1 beta release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1 beta gene expression as well as IL-1 beta secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1 beta release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1 beta mRNA dowregulation followed by suppression of associated caspase-1 activity and IL-1 beta secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1 beta gene expression as well as IL-1 beta secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1 beta signaling pathway might play a priming role in environment-induced DE development. (C) 2015 Elsevier Ltd. All rights reserved.