TLR4 signaling in the pathogenesis of surgical necrotizing enterocolitis

描述（由申请人提供）：本提案的长期目标是了解手术坏死性小肠结肠炎（NEC）的发病机制，NEC是影响压力早产儿的最常见和致命的胃肠道疾病。虽然最初影响的是肠道，但NEC患者可迅速发展为全身性败血症和多系统器官衰竭，并因压倒性的感染性休克而死亡。目前的想法表明，NEC的发展反映了系统性应激导致肠道屏障破坏的影响。这导致细菌和脂多糖（LPS）的易位，这在免疫细胞和肠细胞上被Toll样受体4 （TLR4）识别。我们现在提出，LPS激活TLR4可导致败血症和进一步的肠道损伤，这是NEC的特征。损伤肠黏膜的愈合通常通过上皮修复发生，其中健康的肠细胞向脱落的粘膜迁移。我们已经开发了一种模拟人类NEC的啮齿动物模型，并表明与对照组相比，NEC动物的肠道恢复明显受损。引人注目的是，与野生型幼鼠相比，携带TLR4突变的小鼠NEC的严重程度显著降低。在体外，肠细胞的迁移受RhoA和整合素的调节，用LPS处理肠细胞可以通过激活RhoA和增加细胞-基质粘附而显著抑制肠细胞的迁移。我们现在假设TLR4通过激活RhoA和整合素以及受损的肠道恢复在NEC的发病机制中起关键作用。为了验证这一假设，我们提出：目的1：评估LPS对体外和体内肠细胞中TLR4表达和活性的影响。目标2。确定TLR4信号是否为LPS对RhoA激活和肠细胞迁移的影响所必需，并确定所涉及的信号通路。目标3。通过对RhoA活性和肠细胞迁移的影响，确定TLR4在体内诱导坏死性小肠结肠炎中是否必需。通过验证TLR4信号在NEC发病机制中起关键作用的假设，我们建议了解导致这种毁灭性疾病发展的主要步骤，并为这些脆弱的患者确定新的治疗策略。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the pathogenesis of surgical necrotizing enterocolitis (NEC), the most frequent and lethal gastrointestinal disorder affecting the stressed, preterm infant. Although initially affecting the intestine, patients with NEC may rapidly develop systemic sepsis and multi-system organ failure and death from overwhelming septic shock. Current thinking indicates that the development of NEC reflects the effects of systemic stress causing a breakdown of the intestinal barrier. This leads to the translocation of bacteria and lipopolysaccharide (LPS), which is recognized on immune cells and enterocytes by Toll Like Receptor 4 (TLR4). We now propose that activation of TLR4 by LPS leads to sepsis and further intestinal injury, characteristic of NEC. Healing of the injured intestinal mucosa typically occurs through epithelial restitution, in which healthy enterocytes migrate towards the denuded mucosa. We have developed a rodent model that mimics human NEC, and have shown that intestinal restitution is significantly impaired in animals with NEC compared with controls. Strikingly, mice with mutations in TLR4 have significantly reduced severity of NEC as compared to wild-type littermates. In vitro, enterocyte migration is regulated by RhoA and integrins, and treatment of enterocytes with LPS leads to a significant inhibition in enterocyte migration through the activation of RhoA and increased cell-matrix adhesion. We now hypothesize that TLR4 plays a critical role in the pathogenesis of NEC through the activation of RhoA and integrins and impaired intestinal restitution. To test this hypothesis, we propose: Aim 1 To assess the effects of LPS on the expression and activity of TLR4 in enterocytes in vitro and in vivo. Aim 2.To determine whether TLR4 signaling is necessary for the effects of LPS on RhoA activation and enterocyte migration and to determine the signaling pathways involved. Aim 3.To determine whether TLR4 is required for the induction of necrotizing enterocolitis in vivo through effects on RhoA activity and enterocyte migration. By testing the hypothesis that TLR4 signaling plays a critical role in the pathogenesis of NEC, we propose to understand the principal steps that lead to the development of this devastating illness, and to identify novel treatment strategies for these fragile patients.