Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA; School of Optometry and Ophthalmology, Eye Hospital, Wenzhou Medical University, Wenzhou, China.
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Guangzhou, China.
Ocul Surf. 2022 Oct;26:234-243. doi: 10.1016/j.jtos.2022.10.001. Epub 2022 Oct 6.
To explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease.
Primary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350-500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2-48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy.
The integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress.
Our findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.
探讨天然抗炎细胞因子白细胞介素（IL）37 在预防高渗应激引起的角膜上皮屏障破坏中的新作用和分子机制，高渗应激可发生于干燥性眼病。
从新鲜供体角膜缘外植体培养原代人角膜上皮细胞（HCEC）。通过将 HCEC 从等渗（312mOsM）切换至高渗培养基（350-500mOsM），建立体外干燥模型，建立高渗应激的体外干燥模型，一些细胞用 rhIL-37 或 rhTNF-α 处理不同时间（2-48 小时）。通过 RT-qPCR、ELISA 和共聚焦显微镜免疫荧光染色评估细胞因子和组织蛋白酶 S 的表达以及屏障蛋白的完整性。
高渗培养基暴露的 HCEC 中上皮屏障的完整性明显受到破坏，如紧密连接（TJ、ZO-1、occludin 和 claudin-1）和黏附连接（E-cadherin）蛋白的免疫荧光图像所示。TNF-α加剧了 TJ 屏障功能完整性的高渗诱导破坏，而 IL-37 的暴露则减弱甚至逆转了这些变化。组织蛋白酶 S，由 CTSS 基因编码，被发现直接破坏上皮屏障的完整性。有趣的是，CTSS 表达在 TNF-α和高渗性作用下显著诱导，而外源性 rhIL-37 在高渗应激后抑制 TNF-α和 CTSS 的表达。此外，rhIL-37 恢复了高渗应激下 HCEC 中 2D 和 3D 共聚焦免疫荧光图像中的屏障蛋白完整性。
我们的研究结果表明，抗炎细胞因子 IL-37 通过抑制 TNF-α和 CTSS 的表达，防止高渗应激下角膜上皮屏障破坏的新信号通路。本研究为干燥性眼病保护角膜屏障的机制提供了新的见解。
