Purpose

To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs).

Methods

Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312 mOsM) to hyperosmotic medium (350–500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2–48 h. Some cultures were treated with IL-36α (1–10 ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining.

Results

IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36β and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0 ± 36.33 pg/ml vs 16.79 ± 6.51 pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1β at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1β production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity.

Conclusions

Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.

中文翻译：

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IL-36α/IL-36RA/IL-38 信号介导高渗应激下人角膜上皮细胞的炎症和屏障破坏

目的

使用人角膜上皮细胞 (HCECs) 的体外高渗模型探索 IL-36 细胞因子在干眼病中的不同表达和不同作用。

方法

从新鲜的供体角膜缘外植体培养原代 HCEC。通过单独将 HCEC 从等渗 (312 mOsM) 转换为高渗培养基 (350-500 mOsM) 或添加 rhIL-36RA 或 rhIL-38 2-48 小时来建立高渗模型。一些培养物用 IL-36α (1-10 ng/ml) 处理，有或没有 rhIL-36RA 或 rhIL-38。RT-qPCR检测基因表达；通过ELISA和/或免疫荧光染色评估蛋白质产生和屏障破坏。

结果

IL-36 细胞因子在原代 HCEC 中差异表达。在 3 种促炎激动剂中，IL-36α，但不是 IL-36β 和 IL-36γ，以渗透压依赖性方式被明显诱导，而两种拮抗剂 IL-36RA 和 IL-38 在高渗应激的 HCEC 中被显着抑制。IL-36α 在 mRNA 中增加 4.4 倍，在蛋白质水平增加 6.9 倍（116.0 ± 36.33 pg/ml 对比对照中的 16.79 ± 6.51 pg/ml）450 mOsM，但通过添加 rhIL-36RA 或 rhIL-显着抑制38. 外源性 rhIL-36α 在 mRNA 和蛋白质水平上刺激 TNF-α 和 IL-1β 的表达，并破坏紧密连接蛋白 ZO-1 和 occludin。然而，rhIL-36RA 或 rhIL-38 抑制 TNF-α 和 IL-1β 的产生并保护 HCEC 免受对 IL-36α 或高渗性的反应的屏障破坏。

结论

我们的研究结果表明，用抑制的拮抗剂 IL-36RA 和 IL-38 刺激的促炎性 IL-36α 是高渗性诱导干眼炎症的一种新机制。IL-36RA 和 IL-38 可能在干眼症中具有治疗潜力。