Inflammasome-mediated corneal epithelial cell defenses inhibited by pathogenic bacteria

项目摘要/摘要 健康的角膜上皮是抵御病原菌和环境细菌的有效屏障。因为. 这一点，角膜感染或角膜炎模型通常依赖于通过引入 微生物直接进入角膜基质，在那里它们启动免疫细胞反应，从而破坏组织 并造成威胁视力的疤痕。这种实验方法的一个局限性是上皮防御 机制无法确定或调查。致病菌铜绿假单胞菌是 最常见的感染与戴软性隐形眼镜有关，这表明它是唯一能够引起 即使在上皮完好的情况下，间质也会受到感染。铜绿假单胞菌殖民的一种方式 上皮是通过在上皮细胞内侵入和复制，这已经在培养细胞中进行了研究， 并在小鼠角膜感染模型上进行观察。初步数据表明，该菌产生的EXOS毒素。 铜绿假单胞菌抑制caspase-4介导的侵袭性角膜上皮细胞下垂，为 细菌在受保护的小生境中复制。更多数据表明，另一条不同的炎症体途径是 在角膜上皮细胞中也很活跃，并导致细胞因子IL-1β的产生，它也是EXOS 抑制。因此，exos在角膜感染中的一个新作用可能是延长细菌的生态位 在角膜上皮细胞内复制，同时限制招募免疫细胞的细胞因子的分泌。vbl.使用 选择性监测侵袭细胞的成像方法和CRISPR-Cas9操纵角膜上皮细胞的方法 在遗传学方面，我们将回答关于角膜上皮防御和细菌的三个悬而未决的问题 颠覆：1.EXOS如何阻断caspase-4介导的下垂？2.角膜上皮细胞如何 检测和应对铜绿假单胞菌？3.上皮细胞炎性小体激活和 上睑下垂在保护眼睛免受基质中发生角膜炎的作用？圆满完成这些目标 将识别一种新的眼表宿主防御机制，进一步加深我们对 上皮细胞中的炎性小体，并阐明一种独特的破坏性角膜病原体如何能够 克服主机防御。

Project Summary/Abstract The healthy corneal epithelium is an effective barrier to pathogenic and environmental bacteria. Because of this, corneal infection or keratitis models often rely on bypassing the epithelium altogether by introducing microbes directly into the corneal stroma, where they initiate immune cell responses that can damage tissue and cause vision-threatening scarring. A limitation of this experimental approach is that epithelial defense mechanisms are unable to be identified or investigated. The bacterial pathogen Pseudomonas aeruginosa is the most common infection associated with soft contact lens wear, suggesting it is uniquely capable of causing infection in the stroma even when the epithelium remains intact. One way in which P. aeruginosa colonizes the epithelium is by invading and replicating inside epithelial cells, which has been investigated in cultured cells, and observed in mouse corneal infection models. Preliminary data show that the toxin ExoS produced by P. aeruginosa suppresses caspase-4 mediated pyroptosis of invaded corneal epithelial cells, buying time for bacteria to replicate in a protected niche. Additional data indicate that a different inflammasome pathway is also active in corneal epithelial cells, and leads to production of the cytokine IL-1β, which ExoS also suppresses. Therefore, a novel role for ExoS in corneal infection could be to prolong a niche for bacteria to replicate within corneal epithelial cells, while limiting secretion of cytokines that recruit immune cells. Using imaging methods to selectively monitor invaded cells, and CRISPR-Cas9 to manipulate corneal epithelial cells genetically, we will answer three outstanding questions regarding both corneal epithelial defense, and bacterial subversion of it: 1. How does ExoS block caspase-4-mediated pyroptosis? 2. How do corneal epithelial cells detect and respond to P. aeruginosa? 3. What is the significance of epithelial cell inflammasome activation and pyroptosis in protecting the eye from developing keratitis in the stroma? Successful completion of these aims will identify a new mechanism of host defense at the ocular surface, further our understanding of inflammasomes in epithelial cells, and elucidate how a uniquely devastating corneal pathogen is able to overcome host defenses.