Toll-like receptors and bacterial endophthalmitis

描述（由申请方提供）：细菌性眼内炎是穿透性眼损伤和眼内手术（尤其是白内障手术，美国老年人群中最常见的眼科手术）的一种威胁视力的并发症。大约每1000名患者中有3名在白内障手术后发生细菌性眼内炎。由于美国的老年人口预计将急剧增长，白内障手术的数量也将显著增加，导致眼内炎的发病率成比例增加。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的病原体对于最大限度地减少视网膜损伤至关重要。在初步数据中呈现的最近发现揭示了视网膜通过产生先天免疫的介质来响应TLR 2激动剂Pam 3Cys，并且在细菌感染之前玻璃体内注射Pam 3Cys完全防止了C57 BL/6（B6）小鼠中金黄色葡萄球菌（SA）眼内炎的发展。这导致了TLR 2在视网膜对S的先天免疫反应中发挥关键作用的假设。金黄色葡萄球菌和激活和信号通过TLR决定疾病的结果。该建议的目的是阐明TLR 2激活防止SA眼内炎发展的机制。本研究的目的有三：1）研究TLR 2在视网膜抗SA反应中的作用，以及Pam 3Cys预处理对TLR 2信号通路的调节作用。通过评估TLR介导的细胞信号传导和促炎细胞因子/趋化因子的产生，在正常、TLR 2配体和SA攻击的B6小鼠视网膜和培养的视网膜（小胶质细胞、星形胶质细胞、Muller和RPE）细胞中测试先天应答。2）确定保护性视网膜先天免疫的TLR 2配体诱导的刺激的机制。感染前TLR 2的激活主要通过在随后的细菌攻击后下调视网膜中的促炎（Thl）细胞因子和上调抗微生物肽（AMP）来诱导保护机制。TLR 2-/-和MyD 88-/-小鼠将用于确定这些机制是否是TLR 2/MyD 88依赖性的、对其他细菌有活性的、以及在玻璃体内注射相关的SA眼内炎的小鼠模型中有效。3)目的探讨抗菌肽相关肽（CRAMP）在SA眼内炎视网膜先天性反应中的作用。将使用培养的视网膜细胞和CRAMP敲除（Cnlp-/-）小鼠测试参与CRAMP诱导的TLR信号传导途径和CRAMP调节细菌清除和视网膜完整性保护的机制。这些目标的完成应该提供对视网膜对微生物病原体的先天反应的理解的洞察，并且可能导致识别用于预防手术相关性眼内炎的新的治疗靶点。 公共卫生相关性：本研究将使用眼内炎的小鼠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期间出现的毁灭性并发症。鉴于美国人口老龄化和耐药性细菌感染的增加，这项研究至关重要，可能导致开发新的治疗方法来预防和/或治疗眼部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.