Toll-like receptors and bacterial endophthalmitis

描述(申请人提供)：细菌性眼内炎是穿透性眼损伤和眼内手术的一种威胁视力的并发症，特别是白内障手术，这是美国老年人最常见的眼科手术。大约每1000名患者中就有3名在白内障手术后发生细菌性眼内炎。由于美国老年人口预计将大幅增长，白内障手术的数量也将大幅增加，导致眼内炎的发生率成比例增加。视网膜的视觉特性对炎症引起的损伤高度敏感，因此，快速检测和清除入侵病原体对于最大限度地减少视网膜损伤至关重要。在初步数据中提出的最新发现表明，视网膜通过产生天然免疫的介质来对TLR2激动剂Pam3Cys做出反应，在细菌感染之前玻璃体内注射Pam3Cys，完全防止了C57BL/6(B6)小鼠金黄色葡萄球菌(SA)眼内炎的发展。这导致了一种假设，即TLR2在视网膜对金黄色葡萄球菌的先天免疫反应中发挥关键作用，并通过TLR的激活和信号决定疾病结局。本研究的目的是阐明TLR2激活预防SA眼内炎发生的机制。我们提出了三个具体的目标：1)确定TLR2在视网膜对SA的先天反应中的作用，以及Pam3Cys对TLR2信号的调节作用。将在正常的TLR2配体中测试固有反应，SA通过评估TLR介导的细胞信号和促炎细胞因子/趋化因子的产生来攻击B6小鼠视网膜和培养的视网膜细胞(小胶质细胞、星形胶质细胞、Muller和RPE)；2)确定TLR2配体刺激保护性视网膜固有免疫的机制。TLR2在感染前的激活主要通过下调促炎症(Th1)细胞因子和在随后的细菌攻击时上调视网膜中的抗菌肽(AMPs)来诱导保护机制。TLR2-/-和MyD88-/-小鼠将被用来确定这些机制是否依赖TLR2/MyD88，对其他细菌具有活性，并在玻璃体内注射相关SA眼内炎的小鼠模型中有效。3)探讨放线菌素相关抗菌肽(CRIMP)在SA眼内炎视网膜固有反应中的作用。参与抽筋诱导的TLR信号通路以及抽筋调节细菌清除和保持视网膜完整性的机制，将在培养的视网膜细胞和抽筋基因敲除(Cnlp-/-)小鼠中进行测试。这些目标的完成将有助于深入了解视网膜对微生物病原体的固有反应，并可能导致确定预防手术相关眼内炎的新治疗靶点。 公共卫生相关性：这项研究将使用小鼠眼内炎模型和培养的视网膜细胞来研究细菌性眼内炎的发病机制，细菌性眼内炎是眼科手术中出现的一种破坏性并发症。鉴于美国人口老龄化和抗生素耐药性细菌感染的增加，这项研究具有至关重要的意义，并可能导致开发新的疗法来预防和/或治疗眼科手术相关的细菌性眼内炎。

DESCRIPTION (provided by applicant): Bacterial endophthalmitis is a vision-threatening complication of penetrating eye injury and intraocular surgery, notably cataract surgery, the most common ophthalmic procedure performed in older populations in the United States. Approximately, 3 out of 1000 patients develop bacterial endophthalmitis after cataract surgery. As the aged population in the US is expected to grow dramatically, the number of cataract surgeries performed will also increase significantly, resulting in a proportional increase in the incidence of endophthalmitis. The visual properties of the retina are highly sensitive to inflammation-caused damage therefore, a rapid detection and clearance of invading pathogens is critical in minimizing retinal damage. The recent discovery presented in the preliminary data revealed that the retina responds to the TLR2 agonist Pam3Cys by producing the mediators of innate immunity, and that intravitreal injection of Pam3Cys, prior to bacterial infection, completely prevented the development of Staphylococcus aureus (SA) endophthalmitis in C57BL/6 (B6) mice. This leads to the hypothesis that TLR2 plays a critical role in retinal innate immune response to S. aureus and that activation and signaling through TLR determines the disease outcome. The objective of this proposal is to elucidate the mechanisms by which TLR2 activation prevents the development of SA endophthalmitis. Three specific aims are proposed: 1) To determine the role of TLR2 in retinal innate response against SA, and how TLR2 signaling is modulated by Pam3Cys pretreatment. The innate response will be tested in normal, TLR2 ligand, and the SA challenged B6 mouse retinas and cultured retinal (microglia, astrocytes, Muller and RPE) cells by assessing TLR-mediated cell signaling and production of proinflammatory cytokines/chemokines, 2) To determine the mechanisms of TLR2 ligand-induced stimulation of protective retinal innate immunity. Activation of TLR2 prior to infection induces protective mechanisms mainly by down- regulating proinflammatory (Th1) cytokines and up-regulating antimicrobial peptides (AMPs) in the retina upon subsequent bacterial challenge. TLR2-/- and MyD88-/- mice will be used to determine whether these mechanisms are TLR2/MyD88 dependent, active against other bacteria, and are effective in a mouse model of intravitreal injection-associated SA endophthalmitis. 3) To determine the role of cathelicidin related antimicrobial peptide (CRAMP) in retinal innate responses during SA endophthalmitis. The TLR signaling pathways involved in CRAMP induction and mechanisms by which CRAMP modulates bacterial clearance and preservation of retinal integrity, will be tested using cultured retinal cells and CRAMP knockout (Cnlp-/-) mice. Completion of these aims should provide insight into the understanding of the retinal innate response to microbial pathogens, and may lead to the identification of new therapeutic targets for preventing surgery- associated endophthalmitis. PUBLIC HEALTH RELEVANCE: This study will use a mouse model of endophthalmitis and cultured retinal cells to study the mechanisms underlying the pathogenesis of bacterial endophthalmitis, a devastating complication that arises during ocular surgery. In light of an aging US population and increasing antibiotic-resistant bacterial infection, this study is of paramount importance and may lead to the development of new therapeutics to prevent and/or to treat ocular surgery-associated bacterial endophthalmitis.