Pathogenesis of P. aeruginosa corneal infection

描述：该应用的长期目标是了解角膜上皮细胞对铜绿假单胞菌的分子和细胞反应，并开发化疗和免疫治疗干预措施来治疗这种严重的眼部感染。铜绿假单胞菌通过囊性纤维化跨膜传导调节因子(CFTR)进入角膜上皮细胞，这些细胞内细菌能够在组织内存活并避开宿主防御，导致严重的角膜病理。目标1的目标包括探索CFTR-P的作用。宿主趋化因子和细胞因子反应所显示的角膜病理中铜绿假单胞菌的相互作用。使用表达突变或野生型(WT)cftr的同源人角膜上皮细胞系，铜绿假单胞菌诱导和依赖cftr释放与发病和抵抗感染有关的细胞因子，特别是IL-1、IL-6、IL-18和干扰素-γ。已确定的因素在眼睛感染和病理中的作用将在适当的WT和转基因小鼠身上进行测试，这些小鼠使用角膜划痕损伤眼睛感染模型，或在其中该因素与抗体中和的小鼠身上进行测试。铜绿假单胞菌-CFTR的相互作用取决于脂筏的形成，初步数据表明脂筏的破坏可以预防角膜病理并促进细菌从受感染的眼睛中清除。在目标2中，这些脂筏在发病机制中的作用将通过使用WT和CF角膜细胞系以及由于酸性鞘磷脂酶基因中断而无法形成脂筏的小鼠，以及使用环糊精处理的小鼠(破坏脂筏)来进一步评估。环糊精治疗铜绿假单胞菌眼部感染具有很高的疗效，并成为治疗的重要组成部分。最后，一种全人单抗(MAb)已经被开发出来，以对抗铜绿假单胞菌藻酸盐，这是一种保守的细胞表面多糖，在角膜分离株中低水平表达。重要的是，在感染铜绿假单胞菌的小鼠的角膜中检测到藻酸盐的表达，并且单抗对铜绿假单胞菌感染具有高度的保护作用。在目标3中，将在小鼠角膜感染模型中评估单抗对6种不同铜绿假单胞菌的治疗效果，目标是拥有一种对大多数临床分离株有效的单一免疫治疗试剂，作为目前治疗铜绿假单胞菌溃疡性角膜炎的辅助方法。总体而言，在总结这些目标的基础上，基于对宿主-病原体相互作用的细胞和分子研究，铜绿假单胞菌角膜炎的治疗方法的开发应取得重大进展。

DESCRIPTION: The long-term goal of this application is to understand the molecular and cellular responses of corneal epithelial cells to Pseudomonas aeruginosa and develop chemotherapeutic and immunotherapeutic interventions for treatment of this serious eye infection. P. aeruginosa enters corneal epithelial cells via the cystic fibrosis transmembrane conductance regulator (CFTR), and these intracellular bacteria are able to persist in the tissue and avoid host defenses, leading to serious corneal pathology. Goals of aim 1 include exploring the role of the CFTR-P. aeruginosa interactions in corneal pathology as manifest by host chemokine and cytokine responses. Using isogenic human corneal epithelial cell lines expressing either mutant or wild-type (WT) CFTR, P. aeruginosa induced and CFTR-dependent release of cytokines implicated in pathogenesis and resistance to infection, notably IL-1, IL-6, IL-18 and IFN-gamma, will be examined. The role of the identified factors in eye infection and pathology will be tested in appropriate WT and transgenic mice using a corneal scratch-injured eye infection model or in mice in which the factor is neutralized with antibody. P. aeruginosa- CFTR interactions depend on formation of lipid rafts, and preliminary data indicate disruption of rafts prevents corneal pathology and promotes bacterial clearance from infected eyes. In aim 2, the role of these rafts in pathogenesis will be further evaluated using the WT and CF corneal cell lines as well as mice unable to form lipid rafts due to disruption of the acid sphingomyelinase gene, and in mice treated with cyclodextrin, which disrupts lipid rafts. Cyclodextrin treatment of P. aeruginosa eye infections has the potential to be highly efficacious and become an important component of therapy. Finally, a fully human monoclonal antibody (MAb) has been developed to P. aeruginosa alginate a conserved, cell surface polysaccharide that is expressed at low levels by corneal isolates. Importantly, alginate expression is detected n the corneas of P. aeruginosa infected mice and the MAb is highly protective against P. aeruginosa infection. In aim 3, the Mab will be evaluated for therapeutic efficacy against 6 different P. aeruginosa strains in the murine corneal infection model with the goal of having a single immunotherapeutic reagent useful against most clinical isolates as an adjunct to current treatment modalities for P. aeruginosa ulcerative keratitis. Overall at the conclusion of these aims, significant advances in the development of therapies for P. aeruginosa keratitis should become apparent based on use of cellular and molecular studies on host-pathogen interactions.