The Role of PAF and TLR in NEC

描述(申请人提供)：坏死性小肠结肠炎(NEC)是一种突发性和破坏性的肠道炎症性坏死，影响10%的早产儿，体重低于1500克。虽然这一复杂疾病的病因尚不清楚，但有证据表明，肠缺血、配方奶喂养和细菌攻击激活了血小板激活因子(PAF)和其他炎症介质，导致肠道损伤。我们先前NIH资助的项目的结果表明，PAF激活PAF受体会导致肠上皮细胞发生一系列生理和/或病理变化，包括刺激跨上皮电解质转运、细胞质酸化、caspase激活和DNA断裂。此外，内源性细菌刺激炎性级联反应，是新生儿NEC的先决条件。特定的细菌细胞壁产品刺激多种细胞类型上的Toll受体(TLR)，从而导致重大的下游事件。在健康的肠道环境中，粘膜屏障对细菌细胞壁产物的反应很差，部分原因是TLR低表达或不表达。然而，在新生儿早期发育和细菌定植过程中，TLR在肠道中的表达及其对这一过程的调控尚不完全清楚。我们推测，在新生儿细菌定植的发育过程中，伴随着窒息应激，存在肠道TLR-4基因的异常表达，而PAF参与了这种异常上调。在TLR激活后，下游信号启动一系列事件，最终导致肠坏死和NEC的最终共同途径。利用一种新型的新生小鼠NEC模型，该模型将允许测试转基因动物和细胞培养研究，并将提供更多的机制细节，该研究计划旨在1)表征肠道TLR表达的发育调控，并研究TLR在人NEC和新生小鼠模型中的重要性；2)描述PAF诱导人和啮齿动物肠道上皮细胞TLR表达的信号机制；以及3)研究PAF在体内对肠道TLR基因表达和蛋白质合成的作用。这些研究将阐明PAF和TLR信号之间的相互作用，并阐明这些信号通路是否在新生儿NEC的启动中发挥潜在作用。

DESCRIPTION (provided by applicant): Necrotizing enterocolitis (NEC) is an abrupt and devastating inflammatory necrosis of the bowel affecting 10% of premature infants born weighing less than 1500 grams. Although the etiology of this complex disorder is poorly understood, the evidence suggests that intestinal ischemia, formula feeding, and bacterial challenge activates platelet activating factor (PAF) and other inflammatory mediators leading to intestinal injury. Results from our previous NIH funded project have shown that PAF receptor activation by PAF leads to a series of physiological and/or pathological changes in the intestinal epithelium, including the stimulation of transepithelial electrolyte transport, cytoplasmic acidification, caspase activation and DNA fragmentation. In addition, endogenous bacteria stimulate the inflammatory cascade and are a prerequisite for neonatal NEC. Specific bacterial cell wall products stimulate toll receptors (TLR's) on multiple cell types that result in significant downstream events. In a healthy intestinal environment, the mucosal barrier is poorly responsive to bacterial cell wall products, partly because of low or absent TLR expression. Nonetheless, in the neonate during early development and bacterial colonization, intestinal expression of TLR and regulation of this process is incompletely understood. We hypothesize that during the developmental acquisition of neonatal bacterial colonization with the addition of asphyxia stress, there is abnormal intestinal gene expression of TLR-4, and that PAF contributes to this abnormal upregulation. Following TLR activation, downstream signaling initiates a series of events that culminate in the final common pathway of intestinal necrosis and NEC. Utilizing a novel neonatal mouse model of NEC that will allow testing of genetically-altered animals and cell culture studies that will provide additional mechanistic detail, the research plan is designed to 1) characterize the developmental regulation of intestinal TLR expression and study the importance of TLR in human NEC and the neonatal mouse model, 2) delineate the signaling mechanisms responsible for PAF-induced TLR expression in intestinal epithelial cells from humans and rodents, and 3) investigate the role of PAF on intestinal TLR gene expression and protein synthesis in vivo. The proposed studies will elucidate the interactions between PAF and TLR signaling, and clarify whether these pathways play an underlying role in the initiation of neonatal NEC.