TLR4 Signaling in the Pathogenesis of Surgical Necrotizing Enterocolitis

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，NEC是早产儿胃肠道疾病死亡和残疾的主要原因，并确定这种破坏性疾病的新治疗策略。为了实现这些目标，我们现在将重点放在先天性免疫受体Toll样受体-4 （TLR4）上，它已知是脂多糖（LPS）的受体，并且在第一个资助期，我们已经确定它在该疾病的发病机制中起关键作用。我们现在试图确定TLR4激活导致NEC的分子和细胞机制。为此，我们培育了肠上皮上特异性缺乏TLR4的小鼠，与野生型小鼠相比，这些小鼠被发现不受NEC发育的影响。为了了解其中的机制，我们现在证明了TLR4在正常肠上皮分化的调节中发挥了重要的、以前未被认识到的作用。我们还确定TLR4在肠干细胞（ISC’s）上表达，并且在NEC中ISC增殖（肠粘膜愈合能力的关键决定因素）减少。为了确定ISC's上TLR4的激活是否需要ISC's上TLR4的激活，我们产生了ISC's上特异性缺乏TLR4的小鼠，与野生型小鼠相比，TLR4激活后的小鼠增殖完整。我们现在假设，早产儿中不适当的TLR4信号通过损害肠道干细胞的活性和功能导致NEC的发展。为了验证这一假设，我们提出以下三个目标：目的探讨TLR4激活在坏死性小肠结肠炎发病机制中调控肠上皮分化的作用。目标2。目的：探讨发育中的肠道中TLR4不适当激活对肠道炎症诱导的影响。目标3。研究tlr4介导的肠道干细胞增殖丧失在坏死性小肠结肠炎发病机制中的作用，确定恢复肠道干细胞增殖是否可以预防或减轻疾病的严重程度。为了实现这些研究目标，我们培育了肠上皮缺乏或过表达TLR4或ISC缺乏TLR4的小鼠菌株，开发了肠道干细胞分离和培养技术，并成功地将TLR4激动剂引入发育中的小鼠子宫内肠道。这些研究将在将先天免疫系统与肠道发育联系起来方面取得重大的概念进展，将通过解释基于TLR4在肠道干细胞中的作用的早产儿易感性来推进我们对NEC的理解，并将评估新的抗NEC疗法，这些疗法旨在逆转TLR4对肠道干细胞修复过早小肠损伤粘膜的能力的有害影响。

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 accomplish these goals, we now focus on the innate immune receptor Toll like receptor-4 (TLR4), which is known to be the receptor for lipopolysaccharide (LPS), and which in the first funding period, we have identified to play a critical role in the pathogenesis of this disease. We now seek to define the molecular and cellular mechanisms by which TLR4 activation leads to NEC. To do so, we generated mice that lack TLR4 specifically on the intestinal epithelium, which were found to be protected from NEC development as compared with wild-type mice. In seeking to understand the mechanisms involved, we now demonstrate that TLR4 exerts an important and previously unrecognized role in the regulation of normal intestinal epithelial differentiation. We also determined that TLR4 is expressed on the intestinal stem cells (ISC's), and that ISC proliferation - a key determinant of the ability of the intestinal mucosa to heal - is reduced in NEC. To define whether TLR4 activation on ISC's was required for the loss of ISC proliferation, we generated mice that lack TLR4 specifically on the ISC's, which showed intact proliferation after TLR4 activation compared with wild-type mice. We now hypothesize that inappropriate TLR4 signaling in the preterm infant leads to the development of NEC by impairing the activity and function of the intestinal stem cells. To test this hypothesize we propose the following three aims: Aim 1. To investigate the role of TLR4 activation in regulating intestinal epithelial differentiation in the pathogenesis of necrotizing enterocolitis. Aim 2. To determine the effects of inappropriate activation of TLR4 in the developing intestine on the induction of intestinal inflammation. Aim 3. To characterize the role of TLR4-mediated loss of intestinal stem cell proliferation in the pathogenesis of necrotizing enterocolitis, and to determine whether restoring intestinal stem cell proliferation can prevent or attenuate the severity of the disease. To accomplish these research goals, we have generated mice strains that lack or overexpress TLR4 in intestinal epithelium or that lack TLR4 on the ISC's, have developed techniques of intestinal stem cell isolation and culture and have successfully introduced TLR4 agonists into the developing mouse intestine in utero. These studies will make a significant conceptual advance in linking the innate immune system with intestinal development, will advance our understanding of NEC by explaining the susceptibility of the premature infant based on the role of TLR4 in the intestinal stem cells, and will evaluate novel anti-NEC therapies which seek to reverse the deleterious effects of TLR4 on the ability of the intestinal stem cells to repair the injured mucosa of the premature small intestine.