Modulation of TLR4 and TLR9 Signaling in NEC

描述(由申请人提供)：当前提案的长期目标是了解坏死性小肠结肠炎(NEC)的发病机制并设计新的治疗方法。NEC是早产儿胃肠道疾病死亡的主要原因，其特点是粘膜破坏和脂多糖(LPS)通过炎症的肠道转移。我们最近证实，内毒素受体--Toll样受体4(TLR4)--在NEC的发病机制中起着关键作用。内毒素激活TLR4导致肠上皮细胞的凋亡和绒毛丢失增加，并通过抑制肠道的重建和增殖而减少肠道修复，导致粘膜破坏。我们还表明，TLR4突变的小鼠可以免受NEC的发展，从而减少细胞凋亡和促进肠道愈合。这些发现表明，TLR4可能是NEC的一个有用的治疗靶点。在目前的提案中，我们试图通过抑制肠细胞中的TLR4信号通路来探索细菌来源的DNA(CpG-DNA)作为一种新型的NEC治疗剂的潜在作用。为此，我们假设：TLR9与CpG-DNA一起激活导致TLR4介导的信号在肠上皮细胞中的抑制，从而减弱了内毒素介导的肠上皮细胞凋亡的程度，并逆转了对肠上皮细胞增殖和迁移的抑制。我们进一步假设，TLR9激活抑制TLR4信号的机制涉及到网状蛋白介导的TLR4运输的中断。最后，我们假设，通过抑制肠细胞中的TLR4信号，CpG-DNA激活TLR9将通过减轻肠道损伤程度和促进肠道修复来预防和治疗NEC。为了验证这些假说，我们提出了以下特定的目标，这些目标将在各种原代和培养的肠道细胞系以及野生型小鼠和TLR4和TLR9突变的小鼠身上进行测试：目的1.评估TLR9与CpG-DNA激活对TLR4介导的肠道损伤和修复的保护作用。目的：通过改变TLR4在细胞内的分布，探讨CpG-DNA激活TLR9抑制TLR4信号转导的机制。目的3.通过抑制TLR4的激活，了解TLR9的激活在坏死性小肠结肠炎防治中的作用。公共卫生相关性：这项研究与公共卫生的相关性在于，这项工作试图了解坏死性小肠结肠炎的原因并确定新的治疗方法，坏死性小肠结肠炎是新生儿死亡和残疾的主要原因。我们已经证明，当一种名为Toll样受体4(TLR4)的免疫受体在肠道内激活时，NEC就会发生。目前的建议试图测试激活另一种受体--称为“Toll like Receptor 9(TLR9)”，它导致TLR4的抑制，是否可以作为一种新的治疗方法来治疗患有这种毁灭性疾病的婴儿。

DESCRIPTION (provided by applicant): The long term goal of the current proposal is to understand the pathogenesis and to design novel therapeutic approaches for necrotizing enterocolitis (NEC). NEC is the leading cause of death from gastrointestinal disease in preterm infants, and is characterized by mucosal disruption and the translocation of lipopolysaccharide (LPS) across the inflamed intestine. We have recently demonstrated that the LPS receptor - Toll like receptor 4 (TLR4) - plays a critical role in the pathogenesis of NEC. Activation of TLR4 on enterocytes by LPS leads to an increase in enterocyte apoptosis and villus loss, as well as reduced intestinal repair by blocking intestinal restitution and proliferation, leading to mucosal disruption. We have also shown that mice with mutations in TLR4 are protected from the development of NEC, and accordingly demonstrate reduced apoptosis and enhanced intestinal healing. These findings suggest the possibility that TLR4 may be a useful therapeutic target in NEC. In the current proposal, we seek to explore a potential role for bacterial-derived DNA (CpG-DNA) as a novel therapeutic agent for NEC, through its inhibitory effects on TLR4 signaling in enterocytes. To do so, we hypothesize that: TLR9 activation with CpG-DNA leads to an inhibition of TLR4-mediated signaling in enterocytes resulting in attenuation in the extent of LPS-mediated enterocyte apoptosis and a reversal in the inhibition of enterocyte proliferation and migration. We further hypothesize that the mechanism by which TLR9 activation inhibits TLR4 signaling involves a clathrin-mediated disruption in TLR4 trafficking. Finally, we hypothesize that by inhibiting TLR4 signaling in enterocytes, activation of TLR9 with CpG-DNA will prevent and treat NEC by reducing the extent of intestinal injury and enhancing intestinal repair. To test these hypotheses, we propose the following specific aims, which will be tested using a variety of primary and cultured enterocyte lines, and in wild-type mice and those with mutations in TLR4 and TLR9: AIM 1. To assess the protective effects of TLR9 activation with CpG-DNA on TLR4-mediated intestinal injury and repair. AIM 2. To determine the mechanisms by which TLR9 activation with CpG-DNA inhibits TLR4 signaling in enterocytes through modifying the cellular distribution of TLR4. AIM 3. To understand the role of TLR9 activation in the prevention and treatment of necrotizing enterocolitis through the inhibition of TLR4 activation. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is found in the fact that this work seeks to understand the causes and to identify novel therapies for necrotizing enterocolitis, which is a major cause of death and disability in newborn infants. We have shown that NEC develops when an immune receptor - called toll like receptor 4 (TLR4) - becomes activated within the intestine. The current proposal seeks to test whether activating another receptor - called "Toll like receptor 9 (TLR9)", which leads to the inhibition of TLR4, can serve as a novel therapeutic approach for infants with this devastating disorder.