Bacterial Interactions with Corneal Epithelium in Health and Disease

描述(申请人提供)：角膜感染是导致视力丧失的常见原因。角膜上皮是微生物穿透(穿透)的屏障，已被证明表达与天然防御有关的各种分子/结构。由于缺乏合适的模型，体内对微生物穿越上皮细胞产生抵抗的特定因素尚未确定。在之前的资助时期，发展了多种体内和体外的模型和方法，以使能够研究微生物对上皮细胞的穿越。总而言之，这些模型提供了在有或没有潜在混杂体内因素的情况下进行实验的机会，这是检验假设所需的。还开发了新的成像技术，使角膜上皮内的细菌能够在完整的眼睛中成像、定位和量化，而不需要组织解剖、染色或任何其他样本准备/处理。初步数据显示，去除泪液和随后的组织纸印迹(允许荧光素穿透)可以使细菌在体内附着到小鼠角膜上，但附着的细菌不会穿过上皮。因此，额外的上皮防御系统可以防止细菌穿越不包括荧光素的表面屏障。其他体内数据表明，穿透是通过以下方式实现的：1)MyD88基因敲除，2)表面活性蛋白D(SP-D)基因敲除，或3)EGTA处理角膜表面，EGTA是一种钙离子螯合剂，可破坏细胞间的连接。使用人体细胞在体外收集的数据证实MyD88参与(siRNA敲除增加了遍历)。他们还表明，细菌挑战增强了对穿越的防御，而四种抗菌肽中的任何一种的siRNA击倒则降低了防御。需要检验的假设是，MyD88依赖于对浅表损伤后铜绿假单胞菌横穿角膜上皮的防御涉及细菌诱导的表面活性蛋白D(SP-D)、抗菌肽(AMPs)的上调以及通过TLR或IL-IR介导的信号转导的连接完整性。这将在两个目标上得到检验。目的1探讨SP-D、抗菌因子和物理屏障在依赖MyD88抵抗细菌黏附后穿透中的作用。目的2是确定参与调节角膜上皮细胞这种防御的受体和细胞类型。一般的方法是利用MyD88依赖和细菌挑战作为工具来缩小候选参与因素的范围。有待量化的结果包括细菌穿越、抗菌活性、信使核糖核酸和蛋白质表达，以及不同大小的蛋白质、细菌和乳胶珠的共存。 公共卫生相关性：虽然已知角膜和其他上皮细胞表达各种天然防御因子，但哪些因素参与保护上皮免受附着在表面的微生物的侵袭还有待确定。使用新的体内和体外模型和成像技术收集的初步数据表明，特定的效应器和调节器参与了这种防御。这些发现的扩展可能导致预防传染病的新的和生物兼容的策略。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: While corneal and other epithelia are known to express various innate defense factors, those involved in protecting against epithelial traversal by microbes that adhere to the surface are to be determined. Preliminary data collected using novel in vivo and in vitro models and imaging technologies, suggest specific effectors and regulators are involved in this defense. Expansion of these findings could lead to novel and biocompatible strategies for preventing infectious disease.