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是早产婴儿胃肠道疾病死亡的主要原因，其特征是粘膜破坏和脂多糖（LPS）在炎症肠内的易位。我们最近发现脂多糖受体Toll样受体4 （TLR4）在NEC的发病机制中起关键作用。LPS激活肠细胞上的TLR4导致肠细胞凋亡和绒毛丢失增加，并通过阻断肠道恢复和增殖减少肠道修复，导致粘膜破坏。我们还发现，携带TLR4突变的小鼠可以防止NEC的发生，因此可以减少细胞凋亡并增强肠道愈合。这些发现提示TLR4可能是NEC的一个有用的治疗靶点。在目前的提案中，我们试图通过细菌来源的DNA （CpG-DNA）对肠细胞中TLR4信号的抑制作用，探索其作为NEC新型治疗剂的潜在作用。为此，我们假设：用CpG-DNA激活TLR9导致tlr4介导的肠细胞信号被抑制，从而导致lps介导的肠细胞凋亡程度的衰减，并逆转对肠细胞增殖和迁移的抑制。我们进一步假设，TLR9激活抑制TLR4信号传导的机制涉及网格蛋白介导的TLR4运输中断。最后，我们假设通过抑制肠细胞中的TLR4信号，CpG-DNA激活TLR9可以通过减少肠道损伤程度和增强肠道修复来预防和治疗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细胞系，以及野生型小鼠和TLR4和TLR9突变小鼠进行测试：探讨CpG-DNA激活TLR9对tlr4介导的肠道损伤及修复的保护作用。目标2。目的探讨CpG-DNA激活TLR9通过改变TLR4的细胞分布抑制肠细胞中TLR4信号传导的机制。目标3。通过抑制TLR4激活，了解TLR9激活在坏死性小肠结肠炎预防和治疗中的作用。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。我们已经证明，当一种叫做toll样受体4 （TLR4）的免疫受体在肠道内被激活时，NEC就会发生。目前的提案旨在测试是否激活另一种受体——Toll样受体9 (TLR9)，导致TLR4的抑制，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.