Toll-like receptors and bacterial endophthalmitis

描述（由申请人提供）：细菌性眼内炎是一种视力威胁并发症，是穿透性眼损伤和眼内手术，特别是白内障手术，在美国老年人中最常见的眼科手术。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。随着美国老年人口的急剧增长，白内障手术的数量也将显著增加，导致眼内炎的发病率也将成比例增加。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。初步数据显示，视网膜通过产生先天免疫介质对TLR2激动剂Pam3Cys产生应答，并且在细菌感染之前，在C57BL/6 （B6）小鼠的玻璃体内注射Pam3Cys完全阻止了金葡萄球菌（SA）眼内炎的发展。这导致了TLR2在金黄色葡萄球菌视网膜先天免疫应答中起关键作用的假设，并且通过TLR的激活和信号传导决定了疾病的结果。本研究的目的是阐明TLR2激活阻止SA眼内炎发展的机制。我们提出了三个具体目标：1)确定TLR2在视网膜抗SA先天反应中的作用，以及Pam3Cys预处理如何调节TLR2信号。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，将在正常、TLR2配体、SA刺激的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）细胞中检测先天应答。感染前TLR2的激活主要通过在随后的细菌攻击中下调促炎（Th1）细胞因子和上调抗微生物肽（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于TLR2/MyD88，对其他细菌有活性，并在玻璃体内注射相关SA眼内炎小鼠模型中有效。3)探讨抗菌肽相关抗菌肽（CRAMP）在SA眼内炎视网膜先天反应中的作用。TLR信号通路参与CRAMP的诱导，以及CRAMP调节细菌清除和保存视网膜完整性的机制，将通过培养的视网膜细胞和CRAMP敲除（Cnlp-/-）小鼠进行测试。这些目标的完成将有助于了解视网膜对微生物病原体的先天反应，并可能导致确定新的治疗靶点，以预防手术相关的眼内炎。

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.