Inflammasome-mediated corneal epithelial cell defenses inhibited by pathogenic bacteria

致病菌抑制炎症小体介导的角膜上皮细胞防御

• 批准号: 10688090
• 负责人: Abby R Kroken
• 金额: $ 37.73万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688090
• 关键词: ADP Ribose Transferases; ADP ribosylation; Apoptosis; Bacteria; Bypass; CRISPR/Cas technology; Caspase; Cell Line; Cells; Cessation of life; Cicatrix; Clinical; Contact Lenses; Cornea; Corneal Opacity; Corneal Stroma; Cultured Cells; Cytoplasm; Cytoprotection; Data; Defense Mechanisms; Epithelial Cells; Epithelium; Exotoxins; Eye; Eye Infections; Film; Hela Cells; Host Defense; Host Defense Mechanism; Hydrophilic Contact Lenses; Image; Immune; Immune response; Infection; Inflammasome; Inflammatory; Injections; Interleukin-1 beta; Invaded; Keratitis; Knockout Mice; Life Style; Link; Lipopolysaccharides; Lytic; Measures; Mediating; Microbe; Modeling; Molecular; Monitor; Mus; Neutrophil Infiltration; Outcome; Pathogenicity; Pathway interactions; Predisposition; Process; Production; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Publications; Resolution; Role; Site; Surface; Testing; Time; Tissues; Toxin; Transformed Cell Line; Virulence Factors; Vision; Wild Type Mouse; cell type; corneal epithelium; cytokine; experimental study; imaging modality; microbial; mouse model; mutant; novel; ocular surface; pathogen; pathogenic bacteria; prevent; recruit; response

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.

项目概要/摘要 健康的角膜上皮是致病菌和环境细菌的有效屏障。由于 因此，角膜感染或角膜炎模型通常依赖于完全绕过上皮细胞，引入 微生物直接进入角膜基质，在那里它们引发可能损伤组织的免疫细胞反应 并造成威胁视力的疤痕。该实验方法的局限性在于上皮防御 机制无法被识别或调查。细菌病原体是铜绿假单胞菌 与软性隐形眼镜佩戴相关的最常见感染，表明它具有独特的能力 即使上皮保持完整，间质也会发生感染。铜绿假单胞菌定殖的一种方式 上皮是通过侵入上皮细胞内部并在其内复制，这已在培养细胞中进行了研究， 并在小鼠角膜感染模型中观察到。初步数据显示，P. 铜绿假单胞菌抑制 caspase-4 介导的侵袭角膜上皮细胞焦亡，为 细菌在受保护的生态位中复制。额外的数据表明不同的炎症小体途径是 在角膜上皮细胞中也很活跃，并导致细胞因子 IL-1β 的产生，ExoS 也能产生细胞因子 IL-1β 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存环境。 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。使用 选择性监测入侵细胞的成像方法，以及操纵角膜上皮细胞的 CRISPR-Cas9 从遗传学角度，我们将回答有关角膜上皮防御和细菌的三个悬而未决的问题 颠覆它： 1. ExoS如何阻断caspase-4介导的细胞焦亡？ 2. 角膜上皮细胞是怎样做的 检测并响应铜绿假单胞菌？ 3. 上皮细胞炎症小体激活和激活有何意义？ 焦亡可以保护眼睛免受基质角膜炎的影响？顺利完成这些目标 将确定眼表宿主防御的新机制，进一步加深我们对 上皮细胞中的炎症小体，并阐明了一种独特的破坏性角膜病原体如何能够 克服宿主的防御。