Enteric Glia Regulation of Intestinal Epithelial TLR4 Signaling In Necrotizing Enterocolitis

目前提案的目标是开发坏死性小肠结肠炎(NEC)的新疗法 导致早产儿死于胃肠道疾病的主要原因。NEC被认为起源于 一种对细菌在早产儿肠道定植的旺盛炎症反应 是不完全理解的。基于对老鼠和人类的研究，我们之前发现 肠道上的革兰氏阴性细菌脂多糖受体，即Toll样受体4 上皮细胞，是NEC发育所必需的。TLR4活性在早产儿人类和小鼠中较高 肠道与足月肠道相比，反映了TLR4在肠道发育调控中的作用。这个 TLR4随后被定植细菌激活，导致炎症和NEC。 在寻求扩展这些研究的过程中，我们现在将探索抑制TLR4信号的上游通路， 它们变得无拘无束，导致了NEC。我们已经确定早产儿肠道中的肠神经胶质细胞 通过释放脑源性神经营养因子(BDNF)抑制肠道TLR4，而相对缺乏 早产儿肠道中的肠源性神经胶质细胞通过TLR4信号被夸大，易患NEC。为了支持这一点 观察发现，我们培育了三个品系的胶质细胞缺陷小鼠，这些小鼠表现出新生儿肠道上皮细胞的升高 TLR4信号和增加的NEC严重程度可被BDNF逆转，而小鼠和人类NEC 肠上皮神经胶质细胞和脑源性神经营养因子减少，导致TLR4信号增强。肠源性神经胶质细胞丢失依赖于 TLR4对肠神经胶质细胞本身的激活，因为在肠神经胶质细胞上缺乏TLR4的小鼠受到保护 神经胶质细胞丢失和NEC，提示促进肠神经胶质细胞存活的策略可以治疗NEC。令人惊讶的是，一个 文库筛选得到了一类新的分子，我们的先导是“J11”，它增强了肠源性神经胶质细胞的BDNF 在早产的小鼠和人的肠道中释放，并防止实验性的NEC。 根据以上的初步数据，我们假设肠神经胶质细胞发挥了关键作用，但之前 在肠上皮细胞中抑制TLR4的过度信号从而阻止 NEC，这种保护作用是通过释放BDNF来实现的。我们进一步假设 早产儿肠道中肠神经胶质细胞稀少，再加上TLR4诱导的肠神经胶质细胞丢失，导致 上皮性TLR4信号和NEC的过度表达。最后，我们假设我们最近发现的肠道 胶质细胞激活剂“J11”可能代表了一种新的NEC防治策略。 我们将在三个目标上检验这些假说：目标1.确定肠道神经胶质细胞抑制的机制 TLR4信号在NEC发病机制中的作用；目的2.确定TLR4的作用机制 NEC的肠源性神经胶质细胞丢失，并增强肠内神经胶质细胞的作用以预防NEC；目标3.评估“J11”对 NEC对小鼠和仔猪的防治。这些研究有望提供新的治疗方法。 NEC儿童的治疗方法，基于早产儿肠道内神经胶质细胞对TLR4的影响。

The goal of the current proposal is to develop novel treatments for necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal disease in premature infants. NEC is thought to arise from an exuberant inflammatory response to bacterial colonization in the premature intestine through pathways that are incompletely understood. Based on studies in mice and humans, we previously discovered that the receptor for gram negative bacterial lipopolysaccharide, namely toll-like receptor 4 (TLR4), on the intestinal epithelium, is required for NEC development. TLR4 activity is higher in the premature human and mouse intestine compared to the full-term intestine, reflecting TLR4’s role in governing gut development. The subsequent activation of TLR4 by colonizing bacteria leads to inflammation and NEC. In seeking to extend these studies, we will now explore the upstream pathways that restrain TLR4 signaling, and which become untethered leading to NEC. We have determined that the enteric glia in the premature gut inhibit intestinal TLR4 through the release of brain derived neurotrophic factor (BDNF), while the relative lack of enteric glia in the premature bowel predisposes to NEC through exaggerated TLR4 signaling. In support of this observation, we generated three strains of glial-deficient mice that show elevated newborn intestinal epithelial TLR4 signaling and increased NEC severity, which were reversed by BDNF, while mice and humans with NEC show reduced enteric glia and BDNF, leading to increased TLR4 signaling. Enteric glia loss depended on TLR4 activation on the enteric glia themselves, since mice lacking TLR4 on enteric glia were protected from glial loss and NEC, suggesting that strategies which promote enteric glial survival could treat NEC. Strikingly, a library screen yielded a new class of molecules, of which our lead is “J11”, which enhanced enteric glia BDNF release in both the premature mouse and human intestine ex vivo, and prevents experimental NEC. Based upon the above preliminary data, we hypothesize that the enteric glial play a critical, but previously unrecognized, role in restraining the exaggerated signaling of TLR4 in the intestinal epithelium thus preventing NEC, and that this protective effect occurs through the release of BDNF. We further hypothesize that the paucity of enteric glia in the premature gut, in combination with TLR4-induced enteric glial loss, leads to exaggerated epithelial TLR4 signaling and NEC. Finally, we hypothesize that our recently discovered enteric glial activating agent, “J11”, may represent a novel NEC prevention and treatment strategy. We will test these hypotheses in 3 aims: Aim 1. Determine mechanisms by which the enteric glia restrain TLR4 signaling in the intestinal epithelium in the pathogenesis of NEC; Aim 2. Determine mechanisms of enteric glia loss in NEC, and augment enteric glia effects to prevent NEC; Aim 3. Evaluate “J11” for the prevention and treatment of NEC in mice and piglets. These studies promise to offer novel therapeutic approaches for children with NEC, based upon enteric glial effects on TLR4 within the premature intestine.