Bacterial Interactions with Corneal Epithelium in Health and Disease

描述（由申请人提供）：角膜感染是视力丧失的常见原因。角膜上皮是微生物渗透（穿越）的屏障，已显示表达假定参与先天防御的各种分子/结构。由于缺乏合适的模型，尚未确定体内赋予对微生物穿越上皮的抗性的特定因素。在先前的资助期间，开发了多种体内和体外模型和方法，以研究微生物对上皮细胞的穿透。总之，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，其使得能够在完整眼睛中对角膜上皮内的细菌进行成像、定位和定量，而不需要组织解剖、染色或任何其他样品制备/处理。初步数据显示，泪液的去除和随后的薄纸印迹（其允许荧光素渗透）使得细菌能够在体内粘附到小鼠角膜，但是粘附的细菌不会穿过上皮。因此，额外的上皮防御防止细菌穿越排除荧光素的表面屏障。其他体内数据显示，通过以下方式实现穿越：1）MyD 88敲除，2）表面活性蛋白D（SP-D）敲除，或3）用EGTA（一种已知破坏细胞间连接的钙螯合剂）处理角膜表面。在体外使用人细胞收集的数据证实了MyD 88参与（siRNA敲低增加遍历）。他们还表明，对穿越的防御是由细菌的挑战，并通过siRNA敲低任何四种抗菌肽减少。待测试的假设是，在浅表损伤后持续存在的针对铜绿假单胞菌穿越角膜上皮的MyD 88依赖性防御涉及细菌诱导的表面活性蛋白D（SP-D）、抗菌肽（AMP）和经由TLR或IL-IR介导的信号传导的连接完整性的上调。这将在两个目标中得到检验。目的1探讨SP-D、抗菌因子和物理屏障在MyD 88依赖性防御粘附后细菌穿越中的作用。目的2是确定参与调节角膜上皮中这种防御的受体和细胞类型。一般的方法将是利用MyD 88依赖性和细菌挑战作为工具来缩小候选参与因素。待量化的结果包括细菌穿越、抗微生物活性、mRNA和蛋白质表达以及蛋白质、细菌和各种大小的乳胶珠的共定位。 公共卫生相关性：虽然已知角膜和其他上皮细胞表达各种先天防御因子，但那些参与防止粘附在表面上的微生物穿过上皮的防御因子有待确定。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会导致预防传染病的新的和生物相容性的策略。

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.