Role of the IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to Aspergillus fumigatus infection
Role of the IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to Aspergillus fumigatus infection
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IL-33/ST2/p38信号通路在角膜上皮细胞对烟曲霉感染免疫反应中的作用
DOI：
10.18240/ijo.2019.04.04
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发表时间：
2019
影响因子：
1.4
通讯作者：
Guiqiu Zhao
中科院分区：
文献类型：
--
作者：
Jia You;Jing Lin;Yi-Fan Zhou;Xudong Peng;Hong He;Cui Li;Guoqiang Zhu;Xue-Qi Zhao;Guiqiu Zhao
目的：研究烟曲霉（A. fumigatus）对人角膜及角膜上皮细胞（HCECs）白细胞介素（IL）-33（IL-33）表达的影响。研究IL-33/ST 2/p38信号通路在角膜上皮细胞抗A.采用实时定量逆转录聚合酶链反应（qRT-PCR）和Western blot分析法分别检测HCECs和小鼠角膜中IL-33的mRNA和蛋白表达。用免疫组化法检测健康供体和真菌性角膜炎患者角膜标本中IL-33的表达。用灭活的A.不同浓度的烟曲霉菌丝，含或不含重组人IL-33蛋白、可溶性重组ST 2蛋白、特异性ST 2中和抗体或有丝分裂原活化蛋白激酶（MAPK）p38抑制剂SB 203580，用于评估IL-33/ST 2/p38信号传导在促炎细胞因子调节中的表达和激活。通过qRT-PCR和酶联免疫吸附试验（ELISA）测定IL-6和IL-1β的产生水平。用CCK-8法和细胞计数法检测HCECs的增殖情况，真菌性角膜炎患者角膜组织和实验A组小鼠角膜组织中IL-33的表达水平升高。感染的HCECs中，以及在感染A.烟熏。A.烟曲霉在mRNA和蛋白水平上强烈刺激HCECs产生的促炎细胞因子（IL-6和IL-1β）的产生。这种促炎介质的产生由A. IL-33可进一步刺激烟曲霉毒素的释放，可溶性ST 2蛋白或ST 2中和抗体可抑制IL-33对烟曲霉毒素的释放。此外，IL-33天然促进A.可溶性ST 2蛋白可抑制烟曲霉的表达。MAPK p38抑制剂SB 203580也能抑制A.烟毒诱导的促炎细胞因子产生。IL-33处理48 h和72 h可促进HCECs增殖，而可溶性重组人ST 2蛋白可抑制HCECs增殖。烟曲霉毒素升高了人和小鼠角膜和HCEC中的IL-33表达。因此，IL-33/ST 2/p38信号通路可能在增强角膜上皮细胞对A.烟曲霉感染此外，IL-33通过其受体ST 2促进HCECs的细胞增殖。这些发现表明，一种新的自分泌机制，放大真菌诱导的角膜上皮炎症反应，突出了真菌性角膜炎的潜在治疗靶点。
To investigate the expression of interleukin (IL)-33 in the cornea and human corneal epithelial cells (HCECs) exposed to Aspergillus fumigatus (A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection.The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8 (CCK8) assay and cell count.IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection, as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine (IL-6 and IL-1β) production at both the mRNA and protein levels. This production of pro-inflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48h and 72h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein.A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungal-induced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.