TLR4 signaling in the pathogenesis of surgical necrotizing enterocolitis

描述(申请人提供)：本提案的长期目标是了解外科坏死性小肠结肠炎(NEC)的发病机制，NEC是影响应激早产儿的最常见和最致命的胃肠道疾病。虽然最初影响肠道，但NEC患者可能会迅速发展为全身性脓毒症和多系统器官衰竭，并死于压倒性败血症休克。目前的观点表明，NEC的发展反映了导致肠道屏障破坏的全身应激的影响。这导致细菌和脂多糖(LPS)的移位，并通过Toll样受体4(TLR4)识别免疫细胞和肠道细胞。我们现在提出，内毒素激活TLR4会导致败血症和进一步的肠道损伤，这是NEC的特征。损伤的肠粘膜的愈合通常是通过上皮修复，即健康的肠细胞向剥离的粘膜迁移。我们已经建立了一个模仿人类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。值得注意的是，与野生型小鼠相比，携带TLR4突变的小鼠的NEC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，而内毒素通过激活RhoA和增加细胞与基质的黏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，损害肠道重建，在NEC的发病机制中发挥关键作用。为了验证这一假说，我们提出：目的1评价脂多糖对体外和体内肠上皮细胞TLR4表达和活性的影响。目的2.确定TLR4信号通路是否参与了内毒素对RhoA活化和肠细胞迁移的影响，并探讨了相关的信号转导途径。目的3.通过对RhoA活性和肠道细胞迁移的影响，确定TLR4是否参与体内坏死性小肠结肠炎的诱导。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找到新的治疗策略。

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.