Purpose

Dry eye disease (DED) is a common and multifactor-induced autoimmune ocular surface disease. Environmental factors, such as desiccating stress (DS) and hyperosmolarity, affect the corneal epithelium to induce ocular surface inflammation in DED. We aimed to explore the potential mechanisms by which innate immunity and pyroptosis are initiated in the mucosal epithelium in response to environmental stress.

Methods

Experimental dry eye was established in C57BL/6 J mice and genetic mice on the background of C57BL/6 J mice by subcutaneous injection of scopolamine and exposure to a desiccating environment. SDHCEC cell line was subjected to hyperosmolarity stress (450 mOsM). The phenol red thread tear test and corneal epithelial defects evaluation were used as assessments of severity of DED. RNA-sequencing, quantitative real-time PCR, western blotting and immunofluorescence staining were performed in this study.

Results

Loss-of-function studies indicated that genetic deletion of GSDMD alleviates DS-induced corneal epithelium defects, and GSDMD is needed for IL-33 processing. We further found that NLRP12 collaborates with NLRC4 inflammasome to initiate GSDMD-dependent pyroptosis, which requires TLR4-induced caspase-8 (CASP8) activation in the mucosal corneal epithelium in response to DS.

Conclusions

These findings provide compelling evidence that GSDMD-dependent pyroptosis plays a pivotal role in DED. A novel mechanism involving NLRP12 and NLRC4 inflammasomes-induced GSDMD-dependent pyroptosis, accompanied by IL-33 processing is responsible for ocular surface epithelial defects in response to environmental stress. GSDMD is required for IL-33 processing and the subsequent amplification of inflammatory cascades. These findings reveal novel therapeutic targets for treating DED.

中文翻译：

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NLRP12和NLRC4介导的角膜上皮细胞凋亡是由干眼中GSDMD裂解伴随IL-33加工驱动的。

目的

干眼病（DED）是一种常见的多因素诱发的自身免疫性眼表疾病。干燥压力（DS）和渗透压过高等环境因素会影响角膜上皮，从而诱发DED中的眼表炎症。我们旨在探讨潜在的机制，通过这种机制，先天性免疫和发烧在粘膜上皮细胞中响应环境压力而启动。

方法

通过皮下注射东sco碱并将其暴露在干燥环境中，在C57BL / 6 J小鼠和C57BL / 6 J小鼠背景的遗传小鼠中建立实验干眼。SDHCEC细胞系受到高渗压力（450 mOsM）。酚红线撕裂试验和角膜上皮缺损评估用作DED严重程度的评估。在这项研究中进行了RNA测序，实时荧光定量PCR，免疫印迹和免疫荧光染色。

结果

功能丧失的研究表明，GSDMD的基因缺失可缓解DS诱导的角膜上皮缺损，IL-33加工需要GSDMD。我们进一步发现NLRP12与NLRC4炎性小体共同启动GSDMD依赖的烧伤，这需要TLR4诱导的黏膜角膜上皮中的TLR4诱导的caspase-8（CASP8）活化以响应DS。

结论

这些发现提供了令人信服的证据，即依赖GSDMD的细胞凋亡在DED中起关键作用。涉及NLRP12和NLRC4炎性体诱导的GSDMD依赖性焦磷酸化的新机制，伴随IL-33加工，是响应环境压力导致眼表上皮缺陷的原因。IL-33加工和随后的炎症级联反应扩增需要GSDMD。这些发现揭示了用于治疗DED的新型治疗靶标。