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，其特征在于粘膜破坏和脂多糖（LPS）穿过发炎的肠道移位。我们最近证实LPS受体- Toll样受体4（TLR 4）-在NEC的发病机制中起关键作用。LPS激活肠细胞上的TLR 4导致肠细胞凋亡和绒毛损失增加，以及通过阻断肠恢复和增殖减少肠修复，导致粘膜破坏。我们还表明，TLR 4突变的小鼠被保护免于NEC的发展，并因此表现出减少的细胞凋亡和增强的肠道愈合。这些发现表明TLR 4可能是NEC的有用治疗靶点。在目前的建议中，我们试图探索细菌衍生的DNA（CpG-DNA）作为一种新的治疗药物NEC的潜在作用，通过其对TLR 4信号在肠细胞的抑制作用。为此，我们假设：用CpG-DNA激活TLR 9导致肠细胞中TLR 4介导的信号传导的抑制，从而导致LPS介导的肠细胞凋亡的程度的衰减和肠细胞增殖和迁移的抑制的逆转。我们进一步假设TLR 9激活抑制TLR 4信号传导的机制涉及网格蛋白介导的TLR 4运输中断。最后，我们假设通过抑制肠上皮细胞中的TLR 4信号传导，用CpG-DNA激活TLR 9将通过减少肠损伤的程度和增强肠修复来预防和治疗NEC。为了验证这些假设，我们提出了以下具体目标，将使用各种原代和培养的肠上皮细胞系，并在野生型小鼠和TLR 4和TLR 9突变的小鼠中进行测试：AIM 1。评估CpG-DNA激活TLR 9对TLR 4介导的肠损伤和修复的保护作用。AIM 2.确定CpG-DNA激活TLR 9通过改变TLR 4的细胞分布抑制肠细胞中TLR 4信号传导的机制。AIM 3.了解TLR 9活化在通过抑制TLR 4活化防治坏死性小肠结肠炎中的作用。公共卫生相关性：这项研究与公共卫生的相关性在于，这项工作旨在了解坏死性小肠结肠炎的原因并确定新的治疗方法，这是新生儿死亡和残疾的主要原因。我们已经表明，NEC的发展时，免疫受体-称为Toll样受体4（TLR 4）-成为肠内激活。目前的建议旨在测试激活另一种受体-称为“Toll样受体9（TLR 9）”，导致TLR 4的抑制，是否可以作为患有这种毁灭性疾病的婴儿的新治疗方法。

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.