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.

项目总结/摘要 健康的角膜上皮是病原菌和环境细菌的有效屏障。因为 因此，角膜感染或角膜炎模型通常依赖于通过引入 细菌直接进入角膜基质，在那里它们启动免疫细胞反应， 造成威胁视力的疤痕这种实验方法的局限性在于上皮防御 机制无法确定或调查。细菌病原体绿脓杆菌是 最常见的感染与软性接触透镜配戴有关，这表明它是唯一能够引起 即使在上皮保持完整的情况下，基质中也存在感染。铜绿假单胞菌定植于 上皮是通过侵入上皮细胞并在上皮细胞内复制，这已经在培养的细胞中进行了研究， 并在小鼠角膜感染模型中观察到。初步数据显示，由P. 铜绿假单胞菌抑制半胱天冬酶-4介导的侵入角膜上皮细胞的焦亡， 细菌在受保护的生态位中复制。另外的数据表明，不同的炎性小体途径， 在角膜上皮细胞中也有活性，并导致细胞因子IL-1β的产生，ExoS也 抑制。因此，ExoS在角膜感染中的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。使用 选择性监测侵入细胞的成像方法，以及CRISPR-Cas9操纵角膜上皮细胞 从遗传学上讲，我们将回答三个悬而未决的问题，既关于角膜上皮防御， 颠覆它：1. ExoS如何阻断caspase-4介导的细胞凋亡？2.角膜上皮细胞是如何 检测并应对铜绿假单胞菌？3.上皮细胞炎性小体激活和 角膜炎在保护眼睛免受基质角膜炎的发展方面有何作用？圆满完成这些目标 将确定一个新的机制，宿主防御在眼表，进一步了解 上皮细胞中的炎性小体，并阐明了一种独特的破坏性角膜病原体是如何能够 克服宿主的防御