Bacterial Interactions with Corneal Epithelium in Health and Disease

项目摘要/摘要： 角膜感染是导致视力丧失的常见原因。角膜上皮，微生物渗透的屏障 (Traversal)，已被证明表达与天然防御有关的各种分子/结构。到期 由于缺乏合适的模型，体内对微生物穿越上皮产生抵抗的特定因素 还没有确定。在以前的资助期间，体内和体外的多种模型和方法 是为了能够研究微生物对上皮细胞的穿越而开发的。这些模型共同提供了 在有或没有体内潜在混杂因素的情况下进行实验的机会，例如 需要用来检验假设。还开发了新的成像技术，使细菌能够在 在不需要组织的情况下，对完整眼睛的角膜上皮进行成像、定位和量化 解剖、染色或任何其他样品准备/处理。初步数据显示，撕裂的去除 液体和随后的薄纸印迹(允许荧光素穿透)能够实现细菌黏附 能在小鼠角膜内存活，但附着的细菌不会穿过上皮细胞。因此，额外的 上皮防御系统防止细菌越过表面屏障(不包括荧光素)进行穿越。 其他体内数据表明：1)MyD88基因敲除，2)表面活性蛋白D(SP-D) 敲除，或3)用EGTA治疗角膜表面，EGTA是一种已知的扰乱细胞到细胞的钙络合剂 交汇点。使用人体细胞在体外收集的数据证实MyD88参与(siRNA敲除增加 遍历)。他们还表明，细菌攻击增强了对穿越的防御，而减少了 四种抗菌肽中的任何一种的siRNA被敲除。需要检验的假设是MyD88- 表浅后对铜绿假单胞菌横穿角膜上皮的依赖防御 损伤涉及细菌诱导的表面活性蛋白D(SP-D)、抗菌肽的上调 (AMPS)和连接完整性通过TLR或IL-IR介导的信号传导。这将在两个目标上得到检验。目标1 探讨SP-D、抗菌因子和物理屏障在MyD88依赖防御中的作用 防止细菌粘连后的穿透。目标2是确定参与的受体和细胞类型 调节角膜上皮的这种防御。一般的方法将是利用MyD88依赖 而细菌挑战作为缩小候选范围的工具涉及因素。结果有待量化 包括细菌穿越、抗菌活性、mRNA和蛋白质表达以及蛋白质共定位， 各种大小的细菌和乳胶珠。

Project Summary/Abstract: Corneal infection is a common cause of vision loss. The corneal epithelium, a barrier to microbe penetration (traversal), has been shown to express various molecules/structures assumed involved in innate defense. Due to the lack of suitable models, specific factors conferring resistance to microbial traversal of epithelia in vivo have not been determined. In the previously funded period, multiple in vivo and in vitro models and methods were developed to enable epithelial cell traversal by microbes to be studied. Together, the models provide the opportunity to do experiments either in the context of, or without, potentially confounding in vivo factors, as needed to test hypotheses. Novel imaging technologies have also been developed that enable bacteria within the corneal epithelium to be imaged, localized, and quantified in intact eyes without the need for tissue dissection, staining, or any other sample preparation/processing. Preliminary data show that removal of tear fluid and subsequent tissue paper blotting (which allows fluorescein to penetrate) enables bacterial adherence to the mouse cornea in vivo, but adherent bacteria do not traverse through the epithelium. Thus, additional epithelial defenses protect against bacterial traversal beyond the superficial barriers that exclude fluorescein. Other in vivo data show that traversal is enabled by; 1) MyD88 knockout, 2) surfactant protein D (SP-D) knockout, or 3) treatment of the corneal surface with EGTA, a calcium chelator known to disrupt cell-to-cell junctions. Data collected using human cells in vitro confirm MyD88 involvement (siRNA knockdown increases traversal). They also show that defense against traversal is boosted by bacterial challenge, and reduced by siRNA knockdown of any of four antimicrobial peptides. The hypothesis to be tested is that MyD88- dependent defense against P. aeruginosa traversal of the corneal epithelium persisting after superficial injury involves bacterial-induced upregulation of surfactant protein D (SP-D), antimicrobial peptides (AMPs) and junctional integrity via TLR or IL-IR mediated signaling. This will be tested in two aims. Aim 1 is to explore the roles of SP-D, antimicrobial factors, and physical barriers in MyD88-dependent defense against bacterial traversal after adhesion. Aim 2 is to determine the receptors and cell types involved in regulating this defense in the corneal epithelium. The general approach will be to utilize MyD88-dependence and bacterial challenge as tools for narrowing down candidate involved factors. Outcomes to be quantified include bacterial traversal, antimicrobial activity, mRNA and protein expression, and colocalization of proteins, bacteria and latex beads of various sizes.