Enteric Glia Regulation of Intestinal Epithelial TLR4 Signaling In Necrotizing Enterocolitis

目前建议的目标是开发新的治疗坏死性小肠结肠炎（NEC）， 导致早产儿胃肠疾病死亡的主要原因。NEC被认为源于 通过以下途径对早产肠中细菌定植产生旺盛的炎症反应， 并不完全理解。基于对小鼠和人类的研究，我们先前发现， 肠上皮细胞上的革兰氏阴性细菌脂多糖受体，即Toll样受体4（TLR 4）， 上皮细胞，是NEC发展所必需的。TLR 4活性在早产的人类和小鼠中较高， 肠与足月肠相比，反映了TLR 4在控制肠道发育中的作用。的 随后通过定殖细菌激活TLR 4导致炎症和NEC。 为了扩展这些研究，我们现在将探索抑制TLR 4信号传导的上游途径， 而这一切都将导致NEC。我们已经确定早产儿肠道中的肠神经胶质 通过释放脑源性神经营养因子（BDNF）抑制肠道TLR 4，而相对缺乏 早产肠中的肠神经胶质通过过度的TLR 4信号传导而易患NEC。为支持这一 通过观察，我们产生了三种神经胶质缺陷小鼠， TLR 4信号传导和NEC严重程度增加，这被BDNF逆转，而患有NEC的小鼠和人， 显示肠神经胶质细胞和BDNF减少，导致TLR 4信号增加。肠神经胶质细胞损失依赖于 肠神经胶质细胞本身的TLR 4激活，因为肠神经胶质细胞上缺乏TLR 4的小鼠受到保护， 神经胶质细胞损失和NEC，这表明促进肠神经胶质细胞存活的策略可以治疗NEC。引人注目的是， 文库筛选产生了一类新的分子，其中我们的领导是“J11”，它增强了肠神经胶质细胞BDNF 在早产小鼠和人肠中离体释放，并预防实验性NEC。 基于上述初步数据，我们假设肠神经胶质细胞起关键作用，但以前 在抑制肠上皮细胞中TLR 4的过度信号传导中的作用，从而防止 NEC，这种保护作用是通过释放BDNF发生的。我们进一步假设， 早产肠中肠神经胶质的缺乏，与TLR 4诱导的肠神经胶质损失相结合，导致 过度的上皮细胞TLR 4信号传导和NEC。最后，我们假设我们最近发现的肠道 胶质细胞激活剂“J11”可能代表一种新的NEC预防和治疗策略。 我们将在3个目标中测试这些假设：目标1。确定肠神经胶质细胞抑制 肠上皮细胞TLR 4信号在NEC发病机制中的作用;目的2.确定以下机制 NEC中肠神经胶质细胞的损失，并增强肠神经胶质细胞的作用以预防NEC;目的3.评估“J11”， 预防和治疗小鼠和仔猪的NEC。这些研究有望提供新的治疗方法， 基于肠神经胶质细胞对早产肠内TLR 4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.