Modulation of TLR4 and TLR9 Signaling in NEC

描述（由申请人提供）：拟议研究的目标是确定导致坏死性小肠结肠炎（NEC）发展的机制，这是早产儿胃肠道疾病死亡和残疾的主要原因，并确定这种毁灭性疾病的新治疗策略。要做到这一点，我们将探讨细菌内毒素受体Toll样受体4（TLR 4）在NEC的发病机制中的作用，通过其以前未被认识到的影响，诱导内质网（ER）应激的早产儿肠粘膜。ER应激反映了ER腔内积累的错误折叠蛋白的细胞状态，这导致快速凋亡。在之前的资助期间，我们发现肠粘膜中的TLR 4信号传导是NEC发展所必需的，并且NEC的特征在于TLR 4依赖性诱导肠细胞凋亡，导致粘膜损伤。我们还表明，识别细菌DNA（“CpG-DNA”）的TLR 4的同源受体，即TLR 9，通过抑制TLR 4信号传导和减少肠细胞凋亡来减弱NEC。然而，重要的是，TLR 9抑制导致NEC的TLR 4信号通路的机制仍然无法解释。我们现在提供的证据表明，TLR 4激活导致NEC通过增加在新生儿肠上皮细胞内的ER应激，TLR 9激活通过抑制TLR 4介导的ER应激通过细胞内伴侣热休克蛋白70（Hsp 70），防止NEC。引人注目的是，早产小鼠的特征在于肠上皮细胞中ER应激的TLR 4依赖性增加，这一发现也见于人类婴儿。我们现在假设，TLR 4激活新生儿肠粘膜导致NEC的发展，诱导ER应激在肠上皮细胞导致肠细胞凋亡，这可以逆转TLR 9激活通过细胞内伴侣Hsp 70。我们进一步假设，在子宫内调节肠TLR 4和TLR 9在发育中的胎儿可以减少ER应激和防止NEC的发展。我们将在三个具体目标中检验这一假设：目标1。为了研究TLR 4激活在 在坏死性小肠结肠炎发病机制中调节新生肠上皮细胞的ER应激。AIM 2.确定TLR 9活化降低TLR 4诱导的ER应激和NEC严重程度的机制。AIM 3.探讨子宫内调节TLR 4和TLR 9是否能抑制ER应激，预防NEC的发生。这些研究将通过定义TLR 4信号传导如何导致NEC中的肠上皮细胞凋亡和粘膜损伤，并通过解释早产儿对NEC的易感性增加TLR 4诱导的粘膜ER应激，并通过评价基于早产小肠内ER应激衰减的新型抗NEC疗法，来实现重大的概念性进展。 公共卫生相关性：坏死性小肠结肠炎是早产儿胃肠道疾病死亡的主要原因，目前尚无有效的治疗方法。目前的提案旨在通过关注免疫系统的激活如何导致肠道内壁的破坏来了解坏死性小肠结肠炎的原因，并试图扭转这种损害，以便发现这种毁灭性疾病的新疗法。

DESCRIPTION (provided by applicant): The goals of the proposed research are to determine the mechanisms that lead to the development of necrotizing enterocolitis (NEC), which is the leading cause of death and disability from gastrointestinal disease in premature infants, and to determine novel therapeutic strategies for this devastating disorder. To do so, we will explore the role of the bacterial endotoxin receptor Toll like receptor 4 (TLR4) in the pathogenesis of NEC through its previously unrecognized effects on the induction of endoplasmic reticulum (ER) stress in the intestinal mucosa of the premature infant. ER stress reflects a cellular state of accumulated mis-folded proteins within the lumen of the ER, which leads to rapid apoptosis. In the previous funding period, we discovered that TLR4 signaling in the intestinal mucosa is required for NEC development and that NEC is characterized by a TLR4-dependent induction of enterocyte apoptosis leading to mucosal injury. We also showed that the homologous receptor for TLR4, namely TLR9 - which recognizes bacterial DNA ("CpG-DNA") - attenuated NEC through the inhibition of TLR4 signaling and a reduction in enterocyte apoptosis. Importantly however, the mechanisms by which TLR9 inhibited the TLR4 signaling pathways that lead to NEC remained unexplained. We now provide evidence that TLR4 activation leads to NEC through an increase in ER stress within the newborn intestinal epithelium, and that TLR9 activation protects against NEC by inhibiting TLR4- mediated ER stress via the intracellular chaperone heat shock protein 70 (Hsp70). Strikingly, the premature mouse was characterized by a TLR4-dependent increase in ER stress in the intestinal epithelium, a finding also seen in human infants. We now hypothesize that TLR4 activation within the newborn intestinal mucosa leads to the development of NEC by inducing ER stress in the intestinal epithelium leading to enterocyte apoptosis, which can be reversed by TLR9 activation through the intracellular chaperone Hsp70. We further hypothesize that the in-utero regulation of intestinal TLR4 and TLR9 in the developing fetus can reduce ER stress and prevent the development of NEC. We will test this hypothesis in three specific aims: AIM 1. To investigate the role of TLR4 activation in regulating ER stress in the newborn intestinal epithelium in the pathogenesis of necrotizing enterocolitis. AIM 2. To determine the mechanisms by which TLR9 activation reduces TLR4-induced ER stress and NEC severity. AIM 3. To evaluate whether the in utero regulation of TLR4 and TLR9 can inhibit ER stress and prevent the development of NEC. These studies will make a significant conceptual advance by defining how TLR4 signaling leads to enterocyte apoptosis and mucosal injury in NEC, and by explaining the susceptibility of the premature infant to NEC based on increased TLR4-induced mucosal ER stress, and through the evaluation of novel anti-NEC therapies based upon the attenuation of ER stress within the premature small intestine. PUBLIC HEALTH RELEVANCE: Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in premature infants, and for which there exists no effective cure. The current proposal seeks to understand the causes of necrotizing enterocolitis by focusing on how activation of the immune system causes destruction of the lining of the intestine, and seeks to reverse this damage in order to discover novel treatments for this devastating disease.